The Blue Light-Dependent Polyubiquitination and Degradation of Arabidopsis Cryptochrome2 Requires Multiple E3 Ubiquitin Ligases.

PubMed

Liu, Qing; Wang, Qin; Liu, Bin; Wang, Wei; Wang, Xu; Park, Joon; Yang, Zhenming; Du, Xinglin; Bian, Mingdi; Lin, Chentao

2016-10-01

Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Occurrence, leaching, and degradation of Cry1Ab protein from transgenic maize detritus in agricultural streams

DOE PAGES

Griffiths, Natalie A.; Tank, Jennifer L.; Royer, Todd V.; ...

2017-03-15

The insecticidal Cry1Ab protein expressed by transgenic (Bt) maize can enter adjacent water bodies via multiple pathways, but its fate in stream ecosystems is not as well studied as in terrestrial systems. In this study, we used a combination of field sampling and laboratory experiments to examine the occurrence, leaching, and degradation of soluble Cry1Ab protein derived from Bt maize in agricultural streams. We surveyed 11 agricultural streams in northwestern Indiana, USA, on 6 dates that encompassed the growing season, crop harvest, and snowmelt/spring flooding, and detected Cry1Ab protein in the water column and in flowing subsurface tile drains atmore » concentrations of 3–60 ng/L. In a series of laboratory experiments, submerged Bt maize leaves leached Cry1Ab into stream water with 1% of the protein remaining in leaves after 70 d. Laboratory experiments suggested that dissolved Cry1Ab protein degraded rapidly in microcosms containing water-column microorganisms, and light did not enhance breakdown by stimulating assimilatory uptake of the protein by autotrophs. Here, the common detection of Cry1Ab protein in streams sampled across an agricultural landscape, combined with laboratory studies showing rapid leaching and degradation, suggests that Cry1Ab may be pseudo-persistent at the watershed scale due to the multiple input pathways from the surrounding terrestrial environment.« less

Occurrence, leaching, and degradation of Cry1Ab protein from transgenic maize detritus in agricultural streams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffiths, Natalie A.; Tank, Jennifer L.; Royer, Todd V.

The insecticidal Cry1Ab protein expressed by transgenic (Bt) maize can enter adjacent water bodies via multiple pathways, but its fate in stream ecosystems is not as well studied as in terrestrial systems. In this study, we used a combination of field sampling and laboratory experiments to examine the occurrence, leaching, and degradation of soluble Cry1Ab protein derived from Bt maize in agricultural streams. We surveyed 11 agricultural streams in northwestern Indiana, USA, on 6 dates that encompassed the growing season, crop harvest, and snowmelt/spring flooding, and detected Cry1Ab protein in the water column and in flowing subsurface tile drains atmore » concentrations of 3–60 ng/L. In a series of laboratory experiments, submerged Bt maize leaves leached Cry1Ab into stream water with 1% of the protein remaining in leaves after 70 d. Laboratory experiments suggested that dissolved Cry1Ab protein degraded rapidly in microcosms containing water-column microorganisms, and light did not enhance breakdown by stimulating assimilatory uptake of the protein by autotrophs. Here, the common detection of Cry1Ab protein in streams sampled across an agricultural landscape, combined with laboratory studies showing rapid leaching and degradation, suggests that Cry1Ab may be pseudo-persistent at the watershed scale due to the multiple input pathways from the surrounding terrestrial environment.« less

75 FR 57778 - Issuance of an Experimental Use Permit

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-22

...) necessary for its production in Event TC1507 corn. There are 10.213 kilograms (kg) of eCry3.1Ab, 11.14 grams of Cry1Ab, 8,145 grams of Cry34Ab1, 112.1 grams of Cry35Ab1, 280.6 grams of Vip3Aa20, 217.9 grams of mCry3A, and 177.5 grams of Cry1F proteins in 69,473 kg seeds on 7,311 acres (3,830 PIP and 3,481 non...

Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.

PubMed

Ghosh, Gourab; Ganguly, Shreeparna; Purohit, Arnab; Chaudhuri, Rituparna Kundu; Das, Sampa; Chakraborti, Dipankar

2017-07-01

Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T 0 transformants were selected on the basis of PCR and protein expression profile. T 1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T 1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T 2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T 3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T 0 , T 1, and T 2 ). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.

CryJ-LAMP DNA Vaccines for Japanese Red Cedar Allergy Induce Robust Th1-Type Immune Responses in Murine Model

PubMed Central

Connolly, Michael; Marketon, Anthony

2016-01-01

Allergies caused by Japanese Red Cedar (JRC) pollen affect up to a third of Japanese people, necessitating development of an effective therapeutic. We utilized the lysosomal targeting property of lysosomal-associated membrane protein-1 (LAMP-1) to make DNA vaccines that encode LAMP-1 and the sequences of immunodominant allergen CryJ1 or CryJ2 from the JRC pollen. This novel strategy is designed to skew the CD4 T cell responses to the target allergens towards a nonallergenic Th1 response. CryJ1-LAMP and CryJ2-LAMP were administrated to BALB/c mice and antigen-specific Th1-type IgG2a and Th2-type IgG1 antibodies, as well as IgE antibodies, were assayed longitudinally. We also isolated different T cell populations from immunized mice and adoptively transferred them into naïve mice followed by CryJ1/CryJ2 protein boosts. We demonstrated that CryJ-LAMP immunized mice produce high levels of IFN-γ and anti-CryJ1 or anti-CryJ2 IgG2a antibodies and low levels of IgE antibodies, suggesting that a Th1 response was induced. In addition, we found that CD4+ T cells are the immunological effectors of DNA vaccination in this allergy model. Together, our results suggest the CryJ-LAMP Vaccine has a potential as an effective therapeutic for JRC induced allergy by skewing Th1/Th2 responses. PMID:27239481

Expression of Cry1Ac toxin-binding region in Plutella xyllostella cadherin-like receptor and studying their interaction mode by molecular docking and site-directed mutagenesis.

PubMed

Hu, Xiaodan; Zhang, Xiao; Zhong, Jianfeng; Liu, Yuan; Zhang, Cunzheng; Xie, Yajing; Lin, Manman; Xu, Chongxin; Lu, Lina; Zhu, Qing; Liu, Xianjin

2018-05-01

Cadherin-like protein has been identified as the primary Bacillus thuringiensis (Bt) Cry toxin receptor in Lepidoptera pests and plays a key role in Cry toxin insecticidal. In this study, we successfully expressed the putative Cry1Ac toxin-binding region (CR7-CR11) of Plutella xylostella cadherin-like in Escherichia coli BL21 (DE3). The expressed CR7-CR11 fragment showed binding ability to Cry1Ac toxin under denaturing (Ligand blot) and non-denaturing (ELISA) conditions. The three-dimensional structure of CR7-CR11 was constructed by homology modeling. Molecular docking results of CR7-CR11 and Cry1Ac showed that domain II and domain III of Cry1Ac were taking part in binding to CR7-CR11, while CR7-CR8 was the region of CR7-CR11 in interacting with Cry1Ac. The interaction of toxin-receptor complex was found to arise from hydrogen bond and hydrophobic interaction. Through the computer-aided alanine mutation scanning, amino acid residues of Cry1Ac (Met341, Asn442 and Ser486) and CR7-CR11 (Asp32, Arg101 and Arg127) were predicted as the hot spot residues involved in the interaction of the toxin-receptor complex. At last, we verified the importance role of these key amino acid residues by binding assay. These results will lay a foundation for further elucidating the insecticidal mechanism of Cry toxin and enhancing Cry toxin insecticidal activity by molecular modification. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of water management practices on residue decomposition and degradation of Cry1Ac protein from crop-wild Bt rice hybrids and parental lines during winter fallow season.

PubMed

Xiao, Manqiu; Dong, Shanshan; Li, Zhaolei; Tang, Xu; Chen, Yi; Yang, Shengmao; Wu, Chunyan; Ouyang, Dongxin; Fang, Changming; Song, Zhiping

2015-12-01

Rice is the staple diet of over half of the world's population and Bacillus thuringiensis (Bt) rice expressing insecticidal Cry proteins is ready for deployment. An assessment of the potential impact of Bt rice on the soil ecosystem under varied field management practices is urgently required. We used litter bags to assess the residue (leaves, stems and roots) decomposition dynamics of two transgenic rice lines (Kefeng6 and Kefeng8) containing stacked genes from Bt and sck (a modified CpTI gene encoding a cowpea trypsin inhibitor) (Bt/CpTI), a non-transgenic rice near-isoline (Minghui86), wild rice (Oryza rufipogon) and crop-wild Bt rice hybrid under contrasting conditions (drainage or continuous flooding) in the field. No significant difference was detected in the remaining mass, total C and total N among cultivars under aerobic conditions, whereas significant differences in the remaining mass and total C were detected between Kefeng6 and Kefeng8 and Minghui86 under the flooded condition. A higher decomposition rate constant (km) was measured under the flooded condition compared with the aerobic condition for leaf residues, whereas the reverse was observed for root residues. The enzyme-linked immunosorbent assay (ELISA), which was used to monitor the changes in the Cry1Ac protein in Bt rice residues, indicated that (1) the degradation of the Cry1Ac protein under both conditions best fit first-order kinetics, and the predicted DT50 (50% degradation time) of the Cry1Ac protein ranged from 3.6 to 32.5 days; (2) the Cry1Ac protein in the residue degraded relatively faster under aerobic conditions; and (3) by the end of the study (~154 days), the protein was present at a low concentration in the remaining residues under both conditions. The degradation rate constant was negatively correlated with the initial carbon content and positively correlated with the initial Cry1Ac protein concentration, but it was only correlated with the mass decomposition rate constants under the flooded condition. No Cry1Ac protein was detected in the soils surrounding the buried residue. Our results did not reveal any evidence that the stacked genes (Bt/CpTI) or the presence of the Cry1Ac protein influenced the decomposition dynamics of the rice residues. Furthermore, our results suggested that field drainage after residue incorporation would promote Cry1Ac protein degradation. Copyright © 2015 Elsevier Inc. All rights reserved.

Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin.

PubMed

Oppert, Brenda; Dowd, Scot E; Bouffard, Pascal; Li, Lewyn; Conesa, Ana; Lorenzen, Marcé D; Toutges, Michelle; Marshall, Jeremy; Huestis, Diana L; Fabrick, Jeff; Oppert, Cris; Jurat-Fuentes, Juan Luis

2012-01-01

Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor to date. Furthermore, the methods in this study are useful for comparative analyses in organisms lacking a sequenced genome.

Transcriptome Profiling of the Intoxication Response of Tenebrio molitor Larvae to Bacillus thuringiensis Cry3Aa Protoxin

PubMed Central

Oppert, Brenda; Dowd, Scot E.; Bouffard, Pascal; Li, Lewyn; Conesa, Ana; Lorenzen, Marcé D.; Toutges, Michelle; Marshall, Jeremy; Huestis, Diana L.; Fabrick, Jeff; Oppert, Cris; Jurat-Fuentes, Juan Luis

2012-01-01

Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor to date. Furthermore, the methods in this study are useful for comparative analyses in organisms lacking a sequenced genome. PMID:22558093

Safety and long-term immunological effects of CryJ2-LAMP plasmid vaccine in Japanese red cedar atopic subjects: A phase I study.

PubMed

Su, Yan; Romeu-Bonilla, Eliezer; Anagnostou, Athanasia; Fitz-Patrick, David; Hearl, William; Heiland, Teri

2017-12-02

Japanese Red Cedar (JRC) pollen induced allergy affects one third of Japanese and the development of effective therapies remains an unachieved challenge. We designed a DNA vaccine encoding CryJ2 allergen from the JRC pollen and Lysosomal Associated Membrane Protein 1 (LAMP-1) to treat JRC allergy. These Phase IA and IB trials assessed safety and immunological effects of the investigational CryJ2-LAMP DNA vaccine in both non-sensitive and sensitive Japanese expatriates living in Honolulu, Hawaii. In the Phase IA trial, 6 JRC non-sensitive subjects and 9 JRC and/or Mountain Cedar (MC) sensitive subjects were given 4 vaccine doses (each 4mg/1ml) intramuscularly (IM) at 14-day intervals. Nine JRC and/or MC sensitive subjects were given 4 doses (2 mg/0.5 ml) IM at 14-day intervals. The safety and functional biomarkers were followed for 132 d. Following this, 17 of 24 subjects were recruited into the IB trial and received one booster dose (2 mg/0.5 ml) IM approximately 300 d after the first vaccination dose to which they were randomized in the first phase of the trial. All safety endpoints were met and all subjects tolerated CryJ2-LAMP vaccinations well. At the end of the IA trial, 10 out of 12 JRC sensitive and 6 out of 11 MC sensitive subjects experienced skin test negative conversion, possibly related to the CryJ2-LAMP vaccinations. Collectively, these data suggested that the CryJ2-LAMP DNA vaccine is safe and may be immunologically effective in treating JRC induced allergy.

FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

PubMed Central

Marshall, Kathryn E.; Robinson, Errol W.; Hengel, Shawna M.; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation. PMID:22470473

FRET imaging of diatoms expressing a biosilica-localized ribose sensor.

PubMed

Marshall, Kathryn E; Robinson, Errol W; Hengel, Shawna M; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation.

The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis

PubMed Central

Saleem, Faiza; Shakoori, Abdul Rauf

2017-01-01

The Cry (crystal) proteins from Bacillus thuringiensis are known to have toxicity against a variety of insects and have been exploited to control insect pests through transgenic plants and biopesticides. B. thuringiensis SBS BT-1 carrying the cry2 genes was isolated from soil samples in Pakistan. The 2-kb full length cry2Ac gene was cloned, sequenced, and submitted to the EMBL DNA database (Accession No. AM292031). For expression analysis, Escherichia coli DH5α was transformed with the fragment sub-cloned in pET22b expression vector using NdeI and HindIII restriction sites, and later confirmed by restriction endonuclease analysis. To assess the toxicity of Cry2Ac7 protein against lepidopteran and dipteran insects, BL21 (codon plus) strain of E. coli was further transformed with the recombinant plasmid. The 65-kDa protein was expressed in the form of inclusion bodies up to 180 OD units per liter of the medium. Inclusions were washed with a buffer containing 1.5% Triton-X 100 and >90% pure Cry2Ac7 was obtained. The inclusion bodies were dissolved in 50 mM K2CO3 (pH 11.5), dialyzed, and freeze-dried. This freeze-dried protein as well as inclusion bodies were used in bioassays against larvae of Helicoverpa armigera and Musca domestica. The freeze-dried protein was toxic to H. armigera larvae with an LC50 value of 131 ng/mL. However, Cry2Ac7 produced in E. coli did not show any mortality to M. domestica larvae. This is the first report of Cry2Ac protein toxic to H. armigera. PMID:29099767

Diversity of Bacillus thuringiensis strains in the maize and bean phylloplane and their respective soils in Colombia.

PubMed

Jara, S; Maduell, P; Orduz, S

2006-07-01

To evaluate the distribution of Bacillus thuringiensis strains from maize and bean phylloplane and their respective soils. B. thuringiensis was isolated from the phylloplane and soil of maize and bean from three municipalities in Antioquia, Colombia. Ninety six samples of phylloplane and 24 of soil were analyzed. A total of 214 isolates were obtained from 96 phylloplane samples while 59 isolates were recovered from 24 soil samples. Sixty five per cent and 12% of the phylloplane and soil isolates, respectively, showed activity against Spodoptera frugiperda. These isolates contained delta-endotoxin proteins of 57 and 130 kDa. The most toxic isolates against S. frugiperda had the genotype cry1Aa, cry1Ac, cry1B, and cry1D. In contrast, 27% of the phylloplane isolates and 88% of the soil isolates were active against Culex quinquefasciatus and had protein profiles similar to B. thuringiensis serovar. medellin and B. thuringiensis serovar. israelensis. The most active isolates contain cry4 and cry11 genes. The predominant population of B. thuringiensis on the phylloplane harbored the cry1 gene and was active against S. frugiperda, whereas in soil, isolates harboring cry11 gene and active against C. quinquefasciatus were the majority. The predominance of specific B. thuringiensis populations, both on the leaves and in the soil, suggests the presence of selection in B. thuringiensis populations on the studied environment.

Chilo suppressalis and Sesamia inferens display different susceptibility responses to Cry1A insecticidal proteins.

PubMed

Li, Bo; Xu, Yangyang; Han, Cao; Han, Lanzhi; Hou, Maolin; Peng, Yufa

2015-10-01

Chilo suppressalis and Sesamia inferens are important lepidopteran rice pests that occur concurrently in rice-growing areas of China. The development of transgenic rice expressing Cry1A insecticidal proteins has provided a useful strategy for controlling these pests. This study evaluated the baseline susceptibilities of C. suppressalis and S. inferens to Cry1A, as well as their responses to selection with Cry1A. Wide geographic variation in susceptibility was observed across all field populations. Within a given population, the LC50 of both Cry1Ab and Cry1Ac against S. inferens was drastically higher than that of C. suppressalis. Large LC50 differences (74.6-fold) were detected between the two species for Cry1Ab in the Poyang population, while small differences (3.6-fold) were detected for Cry1Ac in the Changsha population. The Cry1Ac LC50 of C. suppressalis and S. inferens increased 8.4- and 4.4-fold after 21 and eight selection generations respectively. Additionally, the estimated realised heritabilities (h(2) ) of Cry1Ac tolerance were 0.11 in C. suppressalis and 0.292 in S. inferens. S. inferens exhibited a significantly lower susceptibility and more rapidly evolved resistance to Cry1A compared with C. suppressalis. Therefore, S. inferens is more likely to evolve increased resistance, which threatens the sustainability of rice expressing Cry1A protein. © 2014 Society of Chemical Industry.

Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

PubMed

Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

2016-01-01

Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis.

Continuous evolution of B. thuringiensis toxins overcomes insect resistance

PubMed Central

Badran, Ahmed H.; Guzov, Victor M.; Huai, Qing; Kemp, Melissa M.; Vishwanath, Prashanth; Kain, Wendy; Nance, Autumn M.; Evdokimov, Artem; Moshiri, Farhad; Turner, Keith H.; Wang, Ping; Malvar, Thomas; Liu, David R.

2016-01-01

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. We developed a phage-assisted continuous evolution (PACE) selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. PMID:27120167

75 FR 51043 - Pesticide Experimental Use Permit; Receipt of Application; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-18

... (vector pCOT1) in event COT102 cotton, Bt Cry1Ac protein and the genetic material necessary for its production (vector PV-GHBK04) in event MON 15985 cotton, and Bt Cry2Ab2 protein and the genetic material necessary for its production (vector PV-GHBK11) in event MON 15985 cotton. The Agency has determined that...

Production and Characterization of Bacillus thuringiensis Cry1Ac-Resistant Cotton Bollworm Helicoverpa zea (Boddie)▿

PubMed Central

Anilkumar, Konasale J.; Rodrigo-Simón, Ana; Ferré, Juan; Pusztai-Carey, Marianne; Sivasupramaniam, Sakuntala; Moar, William J.

2008-01-01

Laboratory-selected Bacillus thuringiensis-resistant colonies are important tools for elucidating B. thuringiensis resistance mechanisms. However, cotton bollworm, Helicoverpa zea, a target pest of transgenic corn and cotton expressing B. thuringiensis Cry1Ac (Bt corn and cotton), has proven difficult to select for stable resistance. Two populations of H. zea (AR and MR), resistant to the B. thuringiensis protein found in all commercial Bt cotton varieties (Cry1Ac), were established by selection with Cry1Ac activated toxin (AR) or MVP II (MR). Cry1Ac toxin reflects the form ingested by H. zea when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance selection and monitoring. The resistance ratio (RR) for AR exceeded 100-fold after 11 generations and has been maintained at this level for nine generations. This is the first report of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations, reaching only an RR of 12. AR was only partially cross-resistant to MVP II, suggesting that MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. zea and that proteases may be involved with resistance. AR was highly cross-resistant to Cry1Ab toxin but only slightly cross-resistant to Cry1Ab expressing corn leaf powder. AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A, and cypermethrin. Toxin-binding assays showed no significant differences, indicating that resistance was not linked to a reduction in binding. These results aid in understanding why this pest has not evolved B. thuringiensis resistance, and highlight the need to choose carefully the form of B. thuringiensis protein used in experiments. PMID:18024681

Identification of a New cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae

PubMed Central

Zhao, Can; Abdelgaffar, Heba M.; Pan, Hongyu; Song, Fuping

2015-01-01

Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with 125I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution. PMID:25795679

Identification of a New cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae.

PubMed

Zhao, Can; Jurat-Fuentes, Juan Luis; Abdelgaffar, Heba M; Pan, Hongyu; Song, Fuping; Zhang, Jie

2015-06-01

Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with (125)I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Shared Midgut Binding Sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests, Ostrinia nubilalis and Spodoptera frugiperda

PubMed Central

Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

2013-01-01

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with 125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case. PMID:23861865

The Two Cryptochrome/Photolyase Family Proteins Fulfill Distinct Roles in DNA Photorepair and Regulation of Conidiation in the Gray Mold Fungus Botrytis cinerea

PubMed Central

Cohrs, Kim C.

2017-01-01

ABSTRACT The plant-pathogenic leotiomycete Botrytis cinerea is known for the strict regulation of its asexual differentiation programs by environmental light conditions. Sclerotia are formed in constant darkness; black/near-UV (NUV) light induces conidiation; and blue light represses both differentiation programs. Sensing of black/NUV light is attributed to proteins of the cryptochrome/photolyase family (CPF). To elucidate the molecular basis of the photoinduction of conidiation, we functionally characterized the two CPF proteins encoded in the genome of B. cinerea as putative positive-acting components. B. cinerea CRY1 (BcCRY1), a cyclobutane pyrimidine dimer (CPD) photolyase, acts as the major enzyme of light-driven DNA repair (photoreactivation) and has no obvious role in signaling. In contrast, BcCRY2, belonging to the cry-DASH proteins, is dispensable for photorepair but performs regulatory functions by repressing conidiation in white and especially black/NUV light. The transcription of bccry1 and bccry2 is induced by light in a White Collar complex (WCC)-dependent manner, but neither light nor the WCC is essential for the repression of conidiation through BcCRY2 when bccry2 is constitutively expressed. Further, BcCRY2 affects the transcript levels of both WCC-induced and WCC-repressed genes, suggesting a signaling function downstream of the WCC. Since both CPF proteins are dispensable for photoinduction by black/NUV light, the origin of this effect remains elusive and may be connected to a yet unknown UV-light-responsive system. IMPORTANCE Botrytis cinerea is an economically important plant pathogen that causes gray mold diseases in a wide variety of plant species, including high-value crops and ornamental flowers. The spread of disease in the field relies on the formation of conidia, a process that is regulated by different light qualities. While this feature has been known for a long time, we are just starting to understand the underlying molecular mechanisms. Conidiation in B. cinerea is induced by black/near-UV light, whose sensing is attributed to the action of cryptochrome/photolyase family (CPF) proteins. Here we report on the distinct functions of two CPF proteins in the photoresponse of B. cinerea. While BcCRY1 acts as the major photolyase in photoprotection, BcCRY2 acts as a cryptochrome with a signaling function in regulating photomorphogenesis (repression of conidiation). PMID:28667107

Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

PubMed

Ribeiro, Thuanne Pires; Arraes, Fabricio Barbosa Monteiro; Lourenço-Tessutti, Isabela Tristan; Silva, Marilia Santos; Lisei-de-Sá, Maria Eugênia; Lucena, Wagner Alexandre; Macedo, Leonardo Lima Pepino; Lima, Janaina Nascimento; Santos Amorim, Regina Maria; Artico, Sinara; Alves-Ferreira, Márcio; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

2017-08-01

Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T 0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2 -ΔΔCt analyses revealed that T 0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T 0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g -1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T 0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T 1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g -1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

The Macrophage Activation Induced by Bacillus thuringiensis Cry1Ac Protoxin Involves ERK1/2 and p38 Pathways and the Interaction with Cell-Surface-HSP70.

PubMed

Rubio-Infante, Nestor; Ilhuicatzi-Alvarado, Damaris; Torres-Martínez, Marilu; Reyes-Grajeda, Juan Pablo; Nava-Acosta, Raúl; González-González, Edith; Moreno-Fierros, Leticia

2018-01-01

Here, we aimed to further characterize the mechanisms involved in protoxin (p) Cry1Ac-induced macrophage activation. We demonstrated that pCry1Ac induces MAPK ERK1/2, p38, and JNK phosphorylation in RAW264.7 macrophages. Because MAPK activation is mainly triggered via ligand-receptor interactions, we focused on the identification of potential pCry1Ac-receptor proteins. Flow cytometry and confocal analysis showed specific saturable pCry1Ac-binding to the macrophage surface and evidenced its internalization via the clathrin-pathway. We performed immunoprecipitation assays and identified by MALDI-TOF-TOF several possible pCry1Ac-binding proteins, such as heat shock proteins (HSPs), vimentin, α-enolase, and actin; whose interaction and presence was confirmed, respectively, by ligand blot and Western blot assays. We also detected cell-surface (cs) pCry1Ac-HSP70 colocalization, so HSP70 was chosen for further characterization. Co-immunoprecipitation with HSP70 antibodies followed by Western blot confirmed the pCry1Ac-HSP70 interaction. Furthermore, pretreatment of RAW264.7 cells with HSP70 antibodies reduced pCry1Ac-induced ERK1 phosphorylation and MCP-1 production; thus suggest the functional participation of csHSP70 in pCry1Ac-induced macrophage activation. csHSP70 also was evaluated in peritoneal-cavity (PerC) macrophages of untreated BALB/c mice, interestingly it was found that the predominant population namely large-peritoneal-macrophages (LPM) displayed csHSP70 + hi. Furthermore, the dynamics of PerC macrophage subsets, LPM, and small-peritoneal macrophages (SPM) were evaluated in response to in vivo pCry1Ac stimuli in presence or not of phenylethynesulfonamide (PES) a functional HSP70 inhibitor. It was found that pCry1Ac increased the proportion of SPM CD11b + F4/80 + lowMHCII + csHSP70 + low and markedly reduced the amount of LPM CD11b + F4/80 + hiMHCII-csHSP70 + hi; while PES, partially suppressed this pCry1Ac-induced effect, further suggesting the participation of HSP70 in macrophage activation process. J. Cell. Biochem. 119: 580-598, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yang; National Institute of Biological Sciences, Beijing 102206; Xiong, Wei

Although a deficiency in CRY1 or CRY2 correlates with a shorter or longer circadian period, the regulation of CRY proteins in the circadian period has not been well studied. In this study, we found that both CRY1 and CRY2 were able to rescue oscillation in CRY null cells and that they displayed different periods. Furthermore, we demonstrated that protein nuclear import rates, not protein stability, regulate the period-length at the cellular level. Co-transfection of CRY1 and CRY2 in various ratios in the same cells gives rise to the predicted period length in a dose-dependent manner. Given the distinct characteristics ofmore » the C-terminal tails of the CRY1 and CRY2 proteins, our study addresses a long-standing hypothesis that the ratio of these two CRY molecules affects the clock period. - Highlights: • Rhythmic CRY2, like CRY1, in the correct CRY1 phase is sufficient to rescue clock oscillation in CRY null cells. • The short-period mammalian CRY2 protein is more stable than the CRY1 protein. • The N-terminal polypeptide of CRY2 contributes to its stability and Per2 repression, but it does not affect the period. • The C-terminal tails of CRYs regulate their protein stability and nuclear import, but the import rate governs the period. • The ratio, rather than the absolute amounts of CRY1 and CRY2 proteins, determines the period in mammalian cells.« less

Food safety assessment of Cry8Ka5 mutant protein using Cry1Ac as a control Bt protein.

PubMed

Farias, Davi Felipe; Viana, Martônio Ponte; Oliveira, Gustavo Ramos; Santos, Vanessa Olinto; Pinto, Clidia Eduarda Moreira; Viana, Daniel Araújo; Vasconcelos, Ilka Maria; Grossi-de-Sa, Maria Fátima; Carvalho, Ana Fontenele Urano

2015-07-01

Cry8Ka5 is a mutant protein from Bacillus thuringiensis (Bt) that has been proposed for developing transgenic plants due to promising activity against coleopterans, like Anthonomus grandis (the major pest of Brazilian cotton culture). Thus, an early food safety assessment of Cry8Ka5 protein could provide valuable information to support its use as a harmless biotechnological tool. This study aimed to evaluate the food safety of Cry8Ka5 protein following the two-tiered approach, based on weights of evidence, proposed by ILSI. Cry1Ac protein was used as a control Bt protein. The history of safe use revealed no convincing hazard reports for Bt pesticides and three-domain Cry proteins. The bioinformatics analysis with the primary amino acids sequence of Cry8Ka5 showed no similarity to any known toxic, antinutritional or allergenic proteins. The mode of action of Cry proteins is well understood and their fine specificity is restricted to insects. Cry8Ka5 and Cry1Ac proteins were rapidly degraded in simulated gastric fluid, but were resistant to simulated intestinal fluid and heat treatment. The LD50 for Cry8Ka5 and Cry1Ac was >5000 mg/kg body weight when administered by gavage in mice. Thus, no expected relevant risks are associated with the consumption of Cry8Ka5 protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Possible Insecticidal Mechanisms Mediated by Immune-Response-Related Cry-Binding Proteins in the Midgut Juice of Plutella xylostella and Spodoptera exigua.

PubMed

Lu, Keyu; Gu, Yuqing; Liu, Xiaoping; Lin, Yi; Yu, Xiao-Qiang

2017-03-15

Cry toxins are insecticidal toxin proteins produced by a spore-forming Gram-positive bacterium Bacillus thuringiensis. Interactions between the Cry toxins and the receptors from midgut brush border membrane vesicles (BBMVs), such as cadherin, alkaline phosphatase, and aminopeptidase, are key steps for the specificity and insecticidal activity of Cry proteins. However, little is known about the midgut juice proteins that may interfere with Cry binding to the receptors. To validate the hypothesis that there exist Cry-binding proteins that can interfere with the insecticidal process of Cry toxins, we applied Cry1Ab1-coupled Sepharose beads to isolate Cry-binding proteins form midgut juice of Plutella xylostella and Spodoptera exigua. Trypsin-like serine proteases and Dorsal were found to be Cry1Ab1-binding proteins in the midgut juice of P. xylostella. Peroxidase-C (POX-C) was found to be the Cry1Ab1-binding protein in the midgut juice of S. exigua. We proposed possible insecticidal mechanisms of Cry1Ab1 mediated by the two immune-related proteins: Dorsal and POX-C. Our results suggested that there exist, in the midgut juice, Cry-binding proteins, which are different from BBMV-specific receptors.

40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

Code of Federal Regulations, 2010 CFR

2010-07-01

... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...

40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

Code of Federal Regulations, 2011 CFR

2011-07-01

... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...

40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

Code of Federal Regulations, 2013 CFR

2013-07-01

... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...

40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

Code of Federal Regulations, 2012 CFR

2012-07-01

... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...

40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

Code of Federal Regulations, 2014 CFR

2014-07-01

... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...

Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome

PubMed Central

Jiang, Jian; Huang, Ying; Shu, Changlong; Soberón, Mario; Bravo, Alejandra; Liu, Chunqing; Song, Fuping; Lai, Jinsheng

2017-01-01

ABSTRACT The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), containing two cry genes (cry8-like and cry8Ga), is toxic to Holotrichia oblita larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMVs) in H. oblita larvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. The results of saturation binding assays demonstrated that Cry8-like bound specifically to binding sites on BBMVs from H. oblita, and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in the midgut from H. oblita larvae were identified by pulldown assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the H. oblita midgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like-binding proteins. Eight Cry8-like-binding proteins were obtained from pulldown assays conducted with BBMVs. The LC-MS/MS data for these proteins were successfully matched with the H. oblita transcriptome, and BLASTX results identified five proteins as serine protease, transferrin-like, uncharacterized protein LOC658236 of Tribolium castaneum, ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase, alkaline phosphatase, and cadherin. IMPORTANCE Holotrichia oblita is one of the main soil-dwelling pests in China. The larvae damage the roots of crops, resulting in significant yield reductions and economic losses. H. oblita is difficult to control, principally due to its soil-dwelling habits. In recent years, some Cry8 toxins from Bacillus thuringiensis were shown to be active against this pest. Study of the mechanism of action of these Cry8 toxins is needed for their effective use in the control of H. oblita and for their future utilization in transgenic plants. Our work provides important basic data and promotes understanding of the insecticidal mechanism of Cry8 proteins against H. oblita larvae. PMID:28389549

40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

Code of Federal Regulations, 2010 CFR

2010-07-01

... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...

40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

Code of Federal Regulations, 2011 CFR

2011-07-01

... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...

40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

Code of Federal Regulations, 2013 CFR

2013-07-01

... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...

40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

Code of Federal Regulations, 2014 CFR

2014-07-01

... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...

40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

Code of Federal Regulations, 2012 CFR

2012-07-01

... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...

Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

PubMed Central

Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

2013-01-01

The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

Bt Proteins Have No Detrimental Effects on Larvae of the Green Lacewing, Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae).

PubMed

Ali, I; Zhang, S; Muhammad, M S; Iqbal, M; Cui, J-J J-J

2018-06-01

Biosafety of a genetically modified crop is required to be assessed prior to its commercialization. For this, a suitable artificial diet was developed and used to establish a dietary exposure test for assessing the toxicity of midgut-active Bt insecticidal proteins on Chrysopa pallens (Rambur). Subsequently, this dietary exposure test was used to evaluate the toxicity of the proteins Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa on C. pallens larvae. Temporal stability, bioactivity, and the intake of the insecticidal proteins were confirmed by enzyme-linked immunosorbent assay and a sensitive-insect bioassay. The life history characteristics, such as survival, pupation, adult emergence, 7-day larval weight, larval developmental time, and emerged male and female fresh weights remained unaffected, when C. pallens were fed the pure artificial diet (negative control) and the artificial diets containing 200 μg/g of each purified protein: Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, or Vip3Aa. On the contrary, all of the life history characteristics of C. pallens larvae were adversely affected when fed artificial diet containing boric acid (positive control). The results demonstrate that diets containing the tested concentrations of Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa have null effects on C. pallens larvae. The outcome indicates that genetically modified crops expressing the tested Bt proteins are safe for the lacewing, C. pallens.

Bt proteins Cry1Ah and Cry2Ab do not affect cotton aphid Aphis gossypii and ladybeetle Propylea japonica

PubMed Central

Zhao, Yao; Zhang, Shuai; Luo, Jun-Yu; Wang, Chun-Yi; Lv, Li-Min; Wang, Xiao-Ping; Cui, Jin-Jie; Lei, Chao-Liang

2016-01-01

Plant varieties expressing the Bt (Bacillus thuringiensis) insecticidal proteins Cry1Ah and Cry2Ab have potential commercialization prospects in China. However, their potential effects on non-target arthropods (NTAs) remain uncharacterized. The cotton aphid Aphis gossypii is a worldwide pest that damages various important crops. The ladybeetle Propylea japonica is a common and abundant natural enemy in many cropping systems in East Asia. In the present study, the effects of Cry1Ah and Cry2Ab proteins on A. gossypii and P. japonica were assessed from three aspects. First, neither of the Cry proteins affected the growth or developmental characteristics of the two test insects. Second, the expression levels of the detoxification-related genes of the two test insects did not change significantly in either Cry protein treatment. Third, neither of the Cry proteins had a favourable effect on the expression of genes associated with the amino acid metabolism of A. gossypii and the nutrition utilization of P. japonica. In conclusion, the Cry1Ah and Cry2Ab proteins do not appear to affect the cotton aphid A. gossypii or the ladybeetle P. japonica. PMID:26829252

Multitrophic Cry-protein flow in Bt-cotton

USDA-ARS?s Scientific Manuscript database

Although most genetically engineered cotton plants grown today produce the insecticidal Cry-proteins Cry1Ac and Cry2Ab, studies are lacking on multitrophic Cry-protein acquisition in dual-gene cotton fields. Such field data are important for the design and interpretation of laboratory risk assessmen...

Development of monoclonal antibody-based sensitive ELISA for the determination of Cry1Ie protein in transgenic plant.

PubMed

Zhang, Yuwen; Zhang, Wei; Liu, Yan; Wang, Jianhua; Wang, Guoying; Liu, Yunjun

2016-11-01

Cry1Ie is a kind of Bacillus thuringiensis (Bt) toxin protein which has a different action model than the Cry1Ab and Cry1Ac protein. The transgenic maize expressing Cry1Ie might be commercially used in the near future and it is urgent to develop a method to detect Cry1Ie protein in transgenic plants and their products. To develop an ELISA method, Cry1Ie protein was expressed in Escherichia coli strain Transetta DE3, purified with the Ni-NTA spin columns, and then validated by sequencing. Bioassay results showed that the purified Cry1Ie protein was highly toxic to the Asian corn borer. The polyclonal antibody (pAb) and the specific monoclonal antibody (mAb) 1G 4 2D 6 were generated from rabbit and mice which were immunized with Cry1Ie protein, respectively. Western blotting of crude Cry1Ie protein extracts was established by employing mAb 1G 4 2D 6 , whereas the mAb 1G 4 2D 6 negligibly recognized other Bt proteins. Sandwich ELISA against Cry1Ie protein was established by coating with pAb and detecting with mAb 1G 4 2D 6 . The limit of detection (LOD), the limit of quantification (LOQ), and the quantification range of the assay in different matrices of maize plant were determined as 0.27-0.51, 0.29-0.78, and 0.45-15.71 ng/mL, respectively. Recoveries of Cry1Ie protein spiked in different maize tissues ranged from 75.1 to 99.5 %. The established sandwich ELISA was verified using transgenic maize overexpressing Cry1Ie. The results in this study suggested that the established ELISA method is effective for detecting Cry1Ie protein in transgenic plants.

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

PubMed Central

Kelker, Matthew S.; Berry, Colin; Evans, Steven L.; Pai, Reetal; McCaskill, David G.; Wang, Nick X.; Russell, Joshua C.; Baker, Matthew D.; Yang, Cheng; Pflugrath, J. W.; Wade, Matthew; Wess, Tim J.; Narva, Kenneth E.

2014-01-01

Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins. PMID:25390338

Recombinant Cry1Ia protein is highly toxic to cotton boll weevil (Anthonomus grandis Boheman) and fall armyworm (Spodoptera frugiperda).

PubMed

Martins, E S; Aguiar, R W D S; Martins, N F; Melatti, V M; Falcão, R; Gomes, A C M M; Ribeiro, B M; Monnerat, R G

2008-05-01

To evaluate the activity of cry1Ia gene against cotton pests, Spodoptera frugiperda and Anthonomus grandis. Had isolated and characterized a toxin gene from the Bacillus thuringiensis S1451 strain which have been previously shown to be toxic to S. frugiperda and A. grandis. The toxin gene (cry1Ia) was amplified by PCR, sequenced, and cloned into the genome of a baculovirus. The Cry1Ia protein was expressed in baculovirus infected insect cells, producing protein inclusions in infected cells. The Cry1Ia protein has used in bioassays against to S. frugiperda and A. grandis. Bioassays using the purified recombinant protein showed high toxicity to S. frugiperda and A. grandis larvae. Molecular modelling of the Cry1Ia protein translated from the DNA sequence obtained in this work, showed that this protein possibly posses a similar structure to the Cry3A protein. Ultrastructural analysis of midgut cells from A. grandis incubated with the Cry1Ia toxin, showed loss of microvilli integrity. The results indicate that the cry1Ia is a good candidate for the construction of transgenic plants resistant to these important cotton pests.

Molecular and Insecticidal Characterization of a Novel Cry-Related Protein from Bacillus Thuringiensis Toxic against Myzus persicae

PubMed Central

Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Ruiz de Escudero, Iñigo; Caballero, Primitivo

2014-01-01

This study describes the insecticidal activity of a novel Bacillus thuringiensis Cry-related protein with a deduced 799 amino acid sequence (~89 kDa) and ~19% pairwise identity to the 95-kDa-aphidicidal protein (sequence number 204) from patent US 8318900 and ~40% pairwise identity to the cancer cell killing Cry proteins (parasporins Cry41Ab1 and Cry41Aa1), respectively. This novel Cry-related protein contained the five conserved amino acid blocks and the three conserved domains commonly found in 3-domain Cry proteins. The protein exhibited toxic activity against the green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae) with the lowest mean lethal concentration (LC50 = 32.7 μg/mL) reported to date for a given Cry protein and this insect species, whereas it had no lethal toxicity against the Lepidoptera of the family Noctuidae Helicoverpa armigera (Hübner), Mamestra brassicae (L.), Spodoptera exigua (Hübner), S. frugiperda (J.E. Smith) and S. littoralis (Boisduval), at concentrations as high as ~3.5 μg/cm2. This novel Cry-related protein may become a promising environmentally friendly tool for the biological control of M. persicae and possibly also for other sap sucking insect pests. PMID:25384108

Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda

PubMed Central

Lemes, Ana Rita Nunes; Davolos, Camila Chiaradia; Legori, Paula Cristina Brunini Crialesi; Fernandes, Odair Aparecido; Ferré, Juan; Lemos, Manoel Victor Franco; Desiderio, Janete Apparecida

2014-01-01

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested. PMID:25275646

A Western Corn Rootworm Cadherin-like Protein is not Involved in the Binding and Toxicity of Cry34/35Ab1 and Cry3Aa Bacillus Thuringiensis Proteins

USDA-ARS?s Scientific Manuscript database

The western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte is an important insect pest of corn. Bacillus thuringiensis (Bt) insecticidal proteins Cry3Aa (as mCry3A) and Cry34Ab1/Cry35Ab1 have been expressed in transgenic corn and are used to control the insect in the U.S. To date, there ...

Safety Assessment of Bacillus thuringiensis Insecticidal Proteins Cry1C and Cry2A with a Zebrafish Embryotoxicity Test.

PubMed

Gao, Yan-Jie; Zhu, Hao-Jun; Chen, Yi; Li, Yun-He; Peng, Yu-Fa; Chen, Xiu-Ping

2018-05-02

As a result of the large-scale planting of transgenic Bacillus thuringiensis (Bt) crops, fish would be exposed to freely soluble Bt insecticidal protein(s) that are released from Bt crop tissues into adjacent bodies of water or by way of direct feeding on deposited plant material. To assess the safety of two Bt proteins Cry1C and Cry2A to fish, we used zebrafish as a representative species and exposed their embryos to 0.1, 1, and 10 mg/L of the two Cry proteins until 132 h post-fertilization and then several developmental, biochemical, and molecular parameters were evaluated. Chlorpyrifos (CPF), a known toxicant to aquatic organisms, was used as a positive control. Although CPF exposure resulted in significant developmental, biochemical, and molecular changes in the zebrafish embryos, there were almost no significant differences after Cry1C or Cry2A exposure. Thus, we conclude that zebrafish embryos are not sensitive to Cry1C and Cry2A insecticidal proteins at test concentrations.

A Plant Cryptochrome Controls Key Features of the Chlamydomonas Circadian Clock and Its Life Cycle.

PubMed

Müller, Nico; Wenzel, Sandra; Zou, Yong; Künzel, Sandra; Sasso, Severin; Weiß, Daniel; Prager, Katja; Grossman, Arthur; Kottke, Tilman; Mittag, Maria

2017-05-01

Cryptochromes are flavin-binding proteins that act as blue light receptors in bacteria, fungi, plants, and insects and are components of the circadian oscillator in mammals. Animal and plant cryptochromes are evolutionarily divergent, although the unicellular alga Chlamydomonas reinhardtii ( Chlamydomonas throughout) has both an animal-like cryptochrome and a plant cryptochrome (pCRY; formerly designated CPH1). Here, we show that the pCRY protein accumulates at night as part of a complex. Functional characterization of pCRY was performed based on an insertional mutant that expresses only 11% of the wild-type pCRY level. The pcry mutant is defective for central properties of the circadian clock. In the mutant, the period is lengthened significantly, ultimately resulting in arrhythmicity, while blue light-based phase shifts show large deviations from what is observed in wild-type cells. We also show that pCRY is involved in gametogenesis in Chlamydomonas pCRY is down-regulated in pregametes and gametes, and in the pcry mutant, there is altered transcript accumulation under blue light of the strictly light-dependent, gamete-specific gene GAS28 pCRY acts as a negative regulator for the induction of mating ability in the light and for the loss of mating ability in the dark. Moreover, pCRY is necessary for light-dependent germination, during which the zygote undergoes meiosis that gives rise to four vegetative cells. In sum, our data demonstrate that pCRY is a key blue light receptor in Chlamydomonas that is involved in both circadian timing and life cycle progression. © 2017 American Society of Plant Biologists. All Rights Reserved.

Identification of Cha o 3 homolog Cry j 4 from Cryptomeria japonica (Japanese cedar) pollen: Limitation of the present Japanese cedar-specific ASIT.

PubMed

Osada, Toshihiro; Tanaka, Yuki; Yamada, Akira; Sasaki, Eiji; Utsugi, Teruhiro

2018-03-07

About one-third of the Japanese population suffers from Japanese cedar pollinosis, which is frequently accompanied by Japanese cypress pollinosis. Recently, a novel major Japanese cypress pollen allergen, Cha o 3, was discovered. However, whether a Cha o 3 homolog is present in Japanese cedar pollen remains to be determined. Western blot analysis was performed using Cha o 3-specific antiserum. In addition, cloning of the gene encoding Cry j 4 was conducted using total cDNA from the male flower of Japanese cedar trees. Allergen potency and cross-reactivity were investigated using a T-cell proliferation assay, basophil activation test, and ImmunoCAP inhibition assay. A low amount of Cha o 3 homolog protein was detected in Japanese cedar pollen extract. The deduced amino acid sequence of Cry j 4 showed 84% identity to that of Cha o 3. Cross-reactivity between Cry j 4 and Cha o 3 was observed at the T cell and IgE levels. Cry j 4 was discovered as a counterpart allergen of Cha o 3 in Japanese cedar pollen, with a relationship similar to that between Cry j 1-Cha o 1 and Cry j 2-Cha o 2. Our findings also suggest that allergen-specific immunotherapy (ASIT) using Japanese cedar pollen extract does not induce adequate immune tolerance to Cha o 3 due to the low amount of Cry j 4 in Japanese cedar pollen. Therefore, ASIT using Cha o 3 or cypress pollen extract coupled with Japanese cedar pollen extract is required in order to optimally control allergy symptoms during Japanese cypress pollen season. Copyright © 2018 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

Safety assessment of Cry1C protein from genetically modified rice according to the national standards of PR China for a new food resource.

PubMed

Cao, Sishuo; He, Xiaoyun; Xu, Wentao; Ran, Wenjun; Liang, Lixing; Luo, YunBo; Yuan, Yanfang; Zhang, Nan; Zhou, Xin; Huang, Kunlun

2010-12-01

The Cry1C protein produced in Escherichia coli was used for in vitro evaluation and animal studies to support the safety assessment of GM food or feed products containing the Cry1C protein. The Cry1C protein does not have any sequence homology with known allergens or toxins. Although the Cry1C protein was heat stable it was rapidly degraded in vitro with simulated gastric or intestinal fluids. It did not cause adverse effects in mice as administered by gavage at a high level dosage of 5 g (Cry1C protein)/kg body weight. The mutagenicity of this protein was evaluated according to the national standards of People's Republic of China (PR China) for a new food resource. In mutagenic tests, the Cry1C protein caused<4 micronucleated cells per 1000 cells, <16 sperm abnormalities per 1000 cells and was not associated with any increased mutations in the Ames test. Taken together, these data indicate that the Cry1C protein is not a potential allergen or toxin. Copyright © 2010 Elsevier Inc. All rights reserved.

40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...

40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...

40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...

40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...

40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...

[Exposure degree of important non-target arthropods to Cry2Aa in Bt rice fields].

PubMed

Zhang, Qing-Ling; Li, Yun-He; Hua, Hong-Xia; Yang, Chang-Ju; Wu, Hong-Jin; Peng, Yu-Fa

2013-06-01

Based on the principle of "risk = hazard x exposure", the selected representative nontarget organisms in the assessment of the potential effects of insect-resistant genetically modified (GM) crops on non-target arthropods in laboratory are generally the arthropod species highly exposed to the insecticidal proteins expressed by the GM crops in farmland ecosystem. In order to understand the exposure degree of the important arthropod species to Cry proteins in Bt rice fields, and to select the appropriate non-target arthropods in the risk assessment of insect-resistant GM crops, the enzyme-linked immunosorbent assay (ELISA) was conducted to measure the Cry2Aa protein concentration in the arthropods collected from the cry2Aa rice fields at different rice growth stages. The results showed that there was a significant difference in the Cry2Aa content protein concentration in different arthropod species. Some species did not contain Cry2Aa protein, while some species contained larger amounts of Cry2Aa protein. Relative to the arthropods colleted after rice anthesis, the arthropods colleted in rice anthesis contained relative higher concentrations of Cry2Aa protein, especially for the predacious arthropods. No Cry proteins were detected in parasitic arthropods. This study provided references for the laboratory assessment of the effects of GM rice on nontarget arthropods.

Identification of Bacillus thuringiensis Cry1AbMod binding-proteins from Spodoptera frugiperda.

PubMed

Martínez de Castro, Diana L; García-Gómez, Blanca I; Gómez, Isabel; Bravo, Alejandra; Soberón, Mario

2017-12-01

Bacillus thuringiensis Cry toxins are currently used for pest control in transgenic crops but evolution of resistance by the insect pests threatens the use of this technology. The Cry1AbMod toxin was engineered to lack the alpha helix-1 of the parental Cry1Ab toxin and was shown to counter resistance to Cry1Ab and Cry1Ac toxins in different insect species including the fall armyworm Spodoptera frugiperda. In addition, Cry1AbMod showed enhanced toxicity to Cry1Ab-susceptible S. frugiperda populations. To gain insights into the mechanisms of this Cry1AbMod-enhanced toxicity, we isolated the Cry1AbMod toxin binding proteins from S. frugiperda brush border membrane vesicles (BBMV), which were identified by pull-down assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS results indicated that Cry1AbMod toxin could bind to four classes of aminopeptidase (N1, N3, N4 y N5) and actin, with the highest amino acid sequence coverage acquired for APN 1 and APN4. In addition to these proteins, we found other proteins not previously described as Cry toxin binding proteins. This is the first report that suggests the interaction between Cry1AbMod and APN in S. frugiperda. Copyright © 2017 Elsevier Inc. All rights reserved.

Susceptibility of northern corn rootworm Diabrotica barberi (Coleoptera: Chrysomelidae) to mCry3A and eCry3.1Ab Bacillus thuringiensis proteins.

PubMed

Oyediran, Isaac O; Matthews, Phillip; Palekar, Narendra; French, Wade; Conville, Jared; Burd, Tony

2016-12-01

The susceptibility of the northern corn rootworm Diabrotica barberi (Smith & Lawrence) to mCry3A and eCry3.1Ab proteins derived from Bacillus thuringiensis (Bt) was determined using a diet bioassay. Northern corn rootworm neonates were exposed to different concentrations of mCry3A and eCry3.1Ab, incorporated into artificial diet. Larval mortality was evaluated after 7 d. The mCry3A and eCry3.1Ab proteins were found to be toxic to the northern corn rootworm larvae. The LC 50 and LC 99 values for mCry3A were 5.13 and 2482.31 μg/mL, respectively. For eCry3.1Ab, the LC 50 and LC 99 values were 0.49 and 213.01 μg/mL. Based on the estimated lethal concentrations, eCry3.1Ab protein was more efficacious to northern corn rootworm larvae than mCry3A. These lethal concentration values will be used as diagnostic doses for routine annual monitoring for change in susceptibility of field collected northern corn rootworm to mCry3A, and eCry3.1Ab toxins. © 2015 Institute of Zoology, Chinese Academy of Sciences.

40 CFR 174.504 - Bacillus thuringiensis Cry1F protein; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry1F protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.504 Bacillus thuringiensis Cry1F protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in the food...

Non-canonical Phototransduction Mediates Synchronization of the Drosophila melanogaster Circadian Clock and Retinal Light Responses.

PubMed

Ogueta, Maite; Hardie, Roger C; Stanewsky, Ralf

2018-06-04

The daily light-dark cycles represent a key signal for synchronizing circadian clocks. Both insects and mammals possess dedicated "circadian" photoreceptors but also utilize the visual system for clock resetting. In Drosophila, circadian clock resetting is achieved by the blue-light photoreceptor cryptochrome (CRY), which is expressed within subsets of the brain clock neurons. In addition, rhodopsin-expressing photoreceptor cells contribute to light synchronization. Light resets the molecular clock by CRY-dependent degradation of the clock protein Timeless (TIM), although in specific subsets of key circadian pacemaker neurons, including the small ventral lateral neurons (s-LNvs), TIM and Period (PER) oscillations can be synchronized by light independent of CRY and canonical visual Rhodopsin phototransduction. Here, we show that at least three of the seven Drosophila rhodopsins can utilize an alternative transduction mechanism involving the same α-subunit of the heterotrimeric G protein operating in canonical visual phototransduction (Gq). Surprisingly, in mutants lacking the canonical phospholipase C-β (PLC-β) encoded by the no receptor potential A (norpA) gene, we uncovered a novel transduction pathway using a different PLC-β encoded by the Plc21C gene. This novel pathway is important for behavioral clock resetting to semi-natural light-dark cycles and mediates light-dependent molecular synchronization within the s-LNv clock neurons. The same pathway appears to be responsible for norpA-independent light responses in the compound eye. We show that Rhodopsin 5 (Rh5) and Rh6, present in the R8 subset of retinal photoreceptor cells, drive both the long-term circadian and rapid light responses in the eye. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

PubMed

Liu, Mingming; Huang, Rong; Weisman, Adam; Yu, Xiaoyang; Lee, Shih-Hui; Chen, Yalu; Huang, Chao; Hu, Senhua; Chen, Xiuhua; Tan, Wenfeng; Liu, Fan; Chen, Hao; Shea, Kenneth J

2018-05-24

We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R 1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280 FRGSAQGIEGS 290 , 368 RRPFNIGINNQQ 379 and 436 FRSGFSNSSVSIIR 449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368 RRPFNIGINNQQ 379 . This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865 NITIHITDTNNK 876 , a specific recognition site in insect receptor Bt-R 1 , to 368 RRPFNIGINNQQ 379 . The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368 RRPFNIGINNQQ 379 . This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

Tools for controlling protein interactions with light

PubMed Central

Tucker, Chandra L.; Vrana, Justin D.; Kennedy, Matthew J.

2014-01-01

Genetically-encoded actuators that allow control of protein-protein interactions with light, termed ‘optical dimerizers’, are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use controlling transcription, protein localization, and protein secretion with light. Additionally, we provide instructions and software for constructing a pulse-controlled LED light device for use in experiments requiring extended light treatments. PMID:25181301

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut.

PubMed

Nakasu, Erich Y T; Firmino, Alexandre A P; Dias, Simoni C; Rocha, Thales L; Ramos, Hudson B; Oliveira, Gustavo R; Lucena, Wagner; Carlini, Célia R; Grossi-de-Sá, Maria Fátima

2010-07-01

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a approximately 120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots. (c) 2010 Elsevier Inc. All rights reserved.

40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1F protein... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...

40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as plant...

40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...

40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...

40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as plant...

40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...

40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1F protein... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...

40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...

40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry1F protein... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...

40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry1F protein... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...

40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...

40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...

40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...

40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as plant...

40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as plant...

40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...

40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...

40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...

A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii[W

PubMed Central

Beel, Benedikt; Prager, Katja; Spexard, Meike; Sasso, Severin; Weiss, Daniel; Müller, Nico; Heinnickel, Mark; Dewez, David; Ikoma, Danielle; Grossman, Arthur R.; Kottke, Tilman; Mittag, Maria

2012-01-01

Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light–activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities. PMID:22773746

Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants.

PubMed

Farias, Davi F; Peijnenburg, Ad A C M; Grossi-de-Sá, Maria F; Carvalho, Ana F U

2015-01-01

Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 μg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods.

Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants

PubMed Central

Farias, Davi F; Peijnenburg, Ad A C M; Grossi-de-Sá, Maria F; Carvalho, Ana F U

2015-01-01

Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 μg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods. PMID:26513483

Laboratory evaluation of transgenic Populus davidiana×Populus bolleana expressing Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3 genes against gypsy moth and fall webworm.

PubMed

Ding, Liping; Chen, Yajuan; Wei, Xiaoli; Ni, Mi; Zhang, Jiewei; Wang, Hongzhi; Zhu, Zhen; Wei, Jianhua

2017-01-01

Transgenic poplar lines 'Shanxin' (Populus davidiana×Populus bolleana) were generated via Agrobacterium-mediated transformation. The transgenic lines carried the expression cassettes of Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3, respectively. The expression levels of the exogenous insect resistance genes in the transgenic lines were determined by Q-PCR and Western blot. Leaves of the transgenic lines were used for insect feeding bioassays on first instar larvae of the gypsy moth (Lymantria dispar) and fall webworm (Hyphantria cunea). At 5 d of feeding, the mean mortalities of larvae feeding on Cry1Ac + SCK and Cry1Ah3 transgenic poplars leaves were 97% and 91%, while mortality on Cry9Aa3 transgenic lines was about 49%. All gypsy moth and fall webworm larvae were killed in 7-9 days after feeding on leaves from Cry1Ac + SCK or Cry1Ah3 transgenic poplars, while all the fall webworm larvae were killed in 11 days and about 80% of gypsy moth larvae were dead in 14 days after feeding on those from Cry9Aa3 transgenic lines. It was concluded that the transgenic lines of Cry1Ac + SCK and Cry1Ah3 were highly toxic to larvae of both insect species while lines with Cry9Aa3 had lower toxicity,and H. cunea larvae are more sensitive to the insecticidal proteins compared to L. dispar. Transgenic poplar lines toxic to L. dispar and H. cunea could be used to provide Lepidoptera pest resistance to selected strains of poplar trees.

Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens.

PubMed

Bel, Yolanda; Sheets, Joel J; Tan, Sek Yee; Narva, Kenneth E; Escriche, Baltasar

2017-06-01

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens ) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125 -iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. Copyright © 2017 Bel et al.

40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...

40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...

40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...

40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...

40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...

Assessment of potential adjuvanticity of Cry proteins.

PubMed

Joshi, Saurabh S; Barnett, Brian; Doerrer, Nancy G; Glenn, Kevin; Herman, Rod A; Herouet-Guicheney, Corinne; Hunst, Penny; Kough, John; Ladics, Gregory S; McClain, Scott; Papineni, Sabitha; Poulsen, Lars K; Rascle, Jean-Baptiste; Tao, Ai-Lin; van Ree, Ronald; Ward, Jason; Bowman, Christal C

2016-08-01

Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cry proteins are expressed at very low levels in GM crops and are unlikely to function as adjuvants. This conclusion is based on critical review of the published literature on the effects of immunomodulation by Cry proteins, the history of safe use of Cry proteins in foods, safety of the Bt donor organisms, and pre-market weight-of-evidence-based safety assessments for GM crops. Copyright © 2016 Elsevier Inc. All rights reserved.

The fate and transport of the Cry1Ab protein in an agricultural field and laboratory aquatic microcosms.

PubMed

Strain, Katherine E; Lydy, Michael J

2015-08-01

Genetically engineered crops expressing insecticidal crystalline proteins derived from Bacillus thuringiensis (Bt), were commercialized almost two decades ago as a means to manage agricultural pests. The Bt proteins are highly specific and only lethal upon ingestion, limiting the scope of toxicity to target insects. However, concern of exposure to non-target organisms and negative public perceptions regarding Bt crops has caused controversy surrounding their use. The objective of this research was to monitor the fate and transport of a Bt protein, Cry1Ab, in a large-scale agricultural field containing maize expressing the Cry1Ab protein and a non-Bt near isoline, and in aquatic microcosms. The highest environmental concentrations of the Cry1Ab protein were found in runoff water and sediment, up to 130ngL(-1) and 143ngg(-1) dry weight, respectively, with the Cry1Ab protein detected in both Bt and non-Bt maize fields. As surface runoff and residual crop debris can transport Bt proteins to waterways adjacent to agricultural fields, a series of laboratory experiments were conducted to determine the potential fate of the Cry1Ab protein under different conditions. The results showed that sediment type and temperature can influence the degradation of the Cry1Ab protein in an aquatic system and that the Cry1Ab protein can persist for up to two months. Although Cry1Ab protein concentrations measured in the field soil indicate little exposure to terrestrial organisms, the consistent input of Bt-contaminated runoff and crop debris into agricultural waterways is relevant to understanding potential consequences to aquatic species. Copyright © 2015 Elsevier Ltd. All rights reserved.

in silico identification of cross affinity towards Cry1Ac pesticidal protein with receptor enzyme in Bos taurus and sequence, structure analysis of crystal proteins for stability.

PubMed

Ebenezer, King Solomon; Nachimuthu, Ramesh; Thiagarajan, Prabha; Velu, Rajesh Kannan

2013-01-01

Any novel protein introduced into the GM crops need to be evaluated for cross affinity on living organisms. Many researchers are currently focusing on the impact of Bacillus thuringiensis cotton on soil and microbial diversity by field experiments. In spite of this, in silico approach might be helpful to elucidate the impact of cry genes. The crystal a protein which was produced by Bt at the time of sporulation has been used as a biological pesticide to target the insectivorous pests like Cry1Ac for Helicoverpa armigera and Cry2Ab for Spodoptera sp. and Heliothis sp. Here, we present the comprehensive in silico analysis of Cry1Ac and Cry2Ab proteins with available in silico tools, databases and docking servers. Molecular docking of Cry1Ac with procarboxypeptidase from Helicoverpa armigera and Cry1Ac with Leucine aminopeptidase from Bos taurus has showed the 125(th) amino acid position to be the preference site of Cry1Ac protein. The structures were compared with each other and it showed 5% of similarity. The cross affinity of this toxin that have confirmed the earlier reports of ill effects of Bt cotton consumed by cattle.

Adsorption of transgenic insecticidal Cry1Ab protein to SiO2. 2. Patch-controlled electrostatic attraction.

PubMed

Madliger, Michael; Sander, Michael; Schwarzenbach, René P

2010-12-01

Adsorption governs the fate of Cry proteins from genetically modified Bt crops in soils. The effect of ionic strength (I) on the adsorption of Cry1Ab (isoelectric point IEP(Cry1Ab) ≈ 6) to negatively charged quartz (SiO(2)) and positively charged poly-L-lysine (PLL) was investigated at pH 5 to 8, using quartz crystal microbalance with dissipation monitoring and optical waveguide lightmode spectroscopy. Cry1Ab adsorbed via positively and negatively charged surface patches to SiO(2) and PLL, respectively. This patch controlled electrostatic attraction (PCEA) explains the observed increase in Cry1Ab adsorption to sorbents that carried the same net charge as the protein (SiO(2) at pH > IEP(Cry1Ab) and PLL at pH < IEP(Cry1Ab)) with decreasing I. In contrast, the adsorption of two reference proteins, BSA and HEWL, with different adsorption mechanism, were little affected by similar changes of I. Consistent with PCEA, Cry1Ab desorption from SiO(2) at pH > IEP(Cry1Ab) increased with increasing I and pH. Weak Cry1Ab-SiO(2) PCEA above pH 7 resulted in reversible, concentration dependent adsorption. Solution depletion experiments showed that PCEA also governed Cry1Ab adsorption to SiO(2) particles at environmentally relevant concentrations (a few ng mL(-1)). These results imply that models describing Cry1Ab adsorption to charged surfaces in soils need to account for the nonuniform surface charge distribution of the protein.

The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans.

PubMed

Luo, Hui; Xiong, Jing; Zhou, Qiaoni; Xia, Liqiu; Yu, Ziquan

2013-12-01

Several families of crystal proteins from Bacillus thuringiensis exhibit nematicidal activity. Cry5B protein, a pore-forming toxin, has been intensively studied yielding many insights into the mode of action of crystal protein at molecular level and pathogenesis of pore-forming toxins. However, little attention was paid to Cry6A, another representative nematicidal crystal protein. Cry6A shares very low homology with Cry5B at amino acid sequence and probably acts in a distinct pathway from Cry5B and even the other main commercial crystal proteins. In the current study, we comprehensively investigated the nematicidal properties of Cry6Aa2 against the free-living soil nematode Caenorhabditis elegans and examined the physical response of C. elegans to Cry6Aa2 attack. Our results indicate that Cry6Aa2 exhibits high lethal activity to C. elegans and could cause detrimental effects on C. elegans, including obviously suppressed growth, decreased brood size, and even abnormal motility. Meanwhile, our study additionally shows that C. elegans could defend against the Cry6Aa2 toxin harmful threat through behavioral defense responses, such as reduced oral uptake and physical avoidance. In general, this study suggests that Cry6Aa2 possesses diverse nematicidal properties, which strongly indicates that Cry6Aa2 is a promising potential candidate of nematicidal agent. Moreover, this study highlights the importance of behavioral responses in defense of C. elegans for survival and demonstrates the key role of crystal protein in the interaction of B. thuringiensis-C. elegans. These findings could shed light on understanding the interaction of C. elegans with B. thuringiensis and provide a perfect model to study the role of pathogenic factor in the interaction of pathogen-host.

The adsorption features between insecticidal crystal protein and nano-Mg(OH)2.

PubMed

Pan, Xiaohong; Xu, Zhangyan; Zheng, Yilin; Huang, Tengzhou; Li, Lan; Chen, Zhi; Rao, Wenhua; Chen, Saili; Hong, Xianxian; Guan, Xiong

2017-12-01

Nano-Mg(OH) 2 , with low biological toxicity, is an ideal nano-carrier for insecticidal protein to improve the bioactivity. In this work, the adsorption features of insecticidal protein by nano-Mg(OH) 2 have been studied. The adsorption capacity could reach as high as 136 mg g -1 , and the adsorption isotherm had been fitted with Langmuir and Freundlich models. Moreover, the adsorption kinetics followed a pseudo-first or -second order rate model, and the adsorption was spontaneous and an exothermic process. However, high temperatures are not suitable for adsorption, which implies that the temperature would be a critical factor during the adsorption process. In addition, FT-IR confirmed that the protein was adsorbed on the nano-Mg(OH) 2 , zeta potential analysis suggested that insecticidal protein was loaded onto the nano-Mg(OH) 2 not by electrostatic adsorption but maybe by intermolecular forces, and circular dichroism spectroscopy of Cry11Aa protein before and after loading with nano-Mg(OH) 2 was changed. The study applied the adsorption information between Cry11Aa and nano-Mg(OH) 2 , which would be useful in the practical application of nano-Mg(OH) 2 as a nano-carrier.

Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera frugiperda) May Affect the Durability of Current Pyramided Bt Maize Hybrids in Brazil

PubMed Central

Bernardi, Daniel; Salmeron, Eloisa; Horikoshi, Renato Jun; Bernardi, Oderlei; Dourado, Patrick Marques; Carvalho, Renato Assis; Martinelli, Samuel; Head, Graham P.; Omoto, Celso

2015-01-01

Genetically modified plants expressing insecticidal proteins from Bacillus thuringiensis (Bt) offer valuable options for managing insect pests with considerable environmental and economic benefits. Despite the benefits provided by Bt crops, the continuous expression of these insecticidal proteins imposes strong selection for resistance in target pest populations. Bt maize (Zea mays) hybrids have been successful in controlling fall armyworm (Spodoptera frugiperda), the main maize pest in Brazil since 2008; however, field-evolved resistance to the protein Cry1F has recently been reported. Therefore it is important to assess the possibility of cross-resistance between Cry1F and other Cry proteins expressed in Bt maize hybrids. In this study, an F2 screen followed by subsequent selection on MON 89034 maize was used to select an S. frugiperda strain (RR) able to survive on the Bt maize event MON 89034, which expresses the Cry1A.105 and Cry2Ab2 proteins. Field-collected insects from maize expressing the Cry1F protein (event TC1507) represented most of the positive (resistance allele-containing) (iso)families found. The RR strain showed high levels of resistance to Cry1F, which apparently also conferred high levels of cross resistance to Cry1A.105 and Cry1Ab, but had only low-level (10-fold) resistance to Cry2Ab2. Life history studies to investigate fitness costs associated with the resistance in RR strain revealed only small reductions in reproductive rate when compared to susceptible and heterozygous strains, but the RR strain produced 32.2% and 28.4% fewer females from each female relative to the SS and RS (pooled) strains, respectively. Consistent with the lack of significant resistance to Cry2Ab2, MON 89034 maize in combination with appropriate management practices continues to provide effective control of S. frugiperda in Brazil. Nevertheless, the occurrence of Cry1F resistance in S. frugiperda across Brazil, and the cross-resistance to Cry1Ab and Cry1A.105, indicates that current Cry1-based maize hybrids face a challenge in managing S. frugiperda in Brazil and highlights the importance of effective insect resistance management for these technologies. PMID:26473961

Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera frugiperda) May Affect the Durability of Current Pyramided Bt Maize Hybrids in Brazil.

PubMed

Bernardi, Daniel; Salmeron, Eloisa; Horikoshi, Renato Jun; Bernardi, Oderlei; Dourado, Patrick Marques; Carvalho, Renato Assis; Martinelli, Samuel; Head, Graham P; Omoto, Celso

2015-01-01

Genetically modified plants expressing insecticidal proteins from Bacillus thuringiensis (Bt) offer valuable options for managing insect pests with considerable environmental and economic benefits. Despite the benefits provided by Bt crops, the continuous expression of these insecticidal proteins imposes strong selection for resistance in target pest populations. Bt maize (Zea mays) hybrids have been successful in controlling fall armyworm (Spodoptera frugiperda), the main maize pest in Brazil since 2008; however, field-evolved resistance to the protein Cry1F has recently been reported. Therefore it is important to assess the possibility of cross-resistance between Cry1F and other Cry proteins expressed in Bt maize hybrids. In this study, an F2 screen followed by subsequent selection on MON 89034 maize was used to select an S. frugiperda strain (RR) able to survive on the Bt maize event MON 89034, which expresses the Cry1A.105 and Cry2Ab2 proteins. Field-collected insects from maize expressing the Cry1F protein (event TC1507) represented most of the positive (resistance allele-containing) (iso)families found. The RR strain showed high levels of resistance to Cry1F, which apparently also conferred high levels of cross resistance to Cry1A.105 and Cry1Ab, but had only low-level (10-fold) resistance to Cry2Ab2. Life history studies to investigate fitness costs associated with the resistance in RR strain revealed only small reductions in reproductive rate when compared to susceptible and heterozygous strains, but the RR strain produced 32.2% and 28.4% fewer females from each female relative to the SS and RS (pooled) strains, respectively. Consistent with the lack of significant resistance to Cry2Ab2, MON 89034 maize in combination with appropriate management practices continues to provide effective control of S. frugiperda in Brazil. Nevertheless, the occurrence of Cry1F resistance in S. frugiperda across Brazil, and the cross-resistance to Cry1Ab and Cry1A.105, indicates that current Cry1-based maize hybrids face a challenge in managing S. frugiperda in Brazil and highlights the importance of effective insect resistance management for these technologies.

Adsorption of transgenic insecticidal Cry1Ab protein to silica particles. Effects on transport and bioactivity.

PubMed

Madliger, Michael; Gasser, Christoph A; Schwarzenbach, René P; Sander, Michael

2011-05-15

Bt crops are genetically modified to be resistant against insect pests by expressing insecticidal Cry proteins. The processes governing the fate and bioavailability of the expressed transgenic Cry proteins in soils are poorly understood. We studied adsorption of Cry1Ab to negatively charged silica (SiO(2)) particles, a major soil constituent and a model for negatively charged mineral surfaces, at pH 5 to 10 and ionic strengths I = 10 mM to 250 mM, both in solution depletion and saturated column transport experiments. Cry1Ab-SiO(2) interactions were dominated by patch-controlled electrostatic attraction (PCEA), as evident from increasing Cry1Ab attraction to SiO(2) with decreasing I at pH at which both Cry1Ab and SiO(2) were net negatively charged. Experimental and modeling evidence is provided that the surface heterogeneity of SiO(2) particles modulated PCEA, leading to a fraction of adsorption sites with slow Cry1Ab desorption kinetics. Desorption rates from these sites increased upon increasing the solution pH. In toxicity bioassays, we demonstrated that Cry1Ab retained insecticidal activity when adsorbed to SiO(2), suggesting high protein conformational stability during adsorption-desorption cycles. Models predicting Cry1A protein adsorption in soils therefore need to account for combined effects of the nonuniform protein surface charge distribution and of sorbent surface heterogeneity.

Enzyme-linked immunosorbent assay detection and bioactivity of Cry1Ab protein fragments

USDA-ARS?s Scientific Manuscript database

Enzyme-linked immunosorbent assay (ELISA) has emerged as the preferred detection method for Cry proteins in environmental matrices. Concerns exist that ELISAs are capable of detecting fragments of Cry proteins, which may lead to an over-estimation of the concentration of these proteins in the enviro...

Enzyme-linked immunosorbent assay detection and bioactivity of Cry1Ab protein fragments.

PubMed

Albright, Vurtice C; Hellmich, Richard L; Coats, Joel R

2016-12-01

The continuing use of transgenic crops has led to an increased interest in the fate of insecticidal crystalline (Cry) proteins in the environment. Enzyme-linked immunosorbent assays (ELISAs) have emerged as the preferred detection method for Cry proteins in environmental matrices. Concerns exist that ELISAs are capable of detecting fragments of Cry proteins, which may lead to an overestimation of the concentration of these proteins in the environment. Five model systems were used to generate fragments of the Cry1Ab protein, which were then analyzed by ELISAs and bioassays. Fragments from 4 of the model systems were not detectable by ELISA and did not retain bioactivity. Fragments from the proteinase K model system were detectable by ELISA and retained bioactivity. In most cases, ELISAs appear to provide an accurate estimation of the amount of Cry proteins in the environment, as detectable fragments retained bioactivity and nondetectable fragments did not retain bioactivity. Environ Toxicol Chem 2016;35:3101-3112. © 2016 SETAC. © 2016 SETAC.

Management of Field-Evolved Resistance to Bt Maize in Argentina: A Multi-Institutional Approach.

PubMed

Signorini, Ana M; Abratti, Gustavo; Grimi, Damián; Machado, Marcos; Bunge, Florencia F; Parody, Betiana; Ramos, Laura; Cortese, Pablo; Vesprini, Facundo; Whelan, Agustina; Araujo, Mónica P; Podworny, Mariano; Cadile, Alejandro; Malacarne, María F

2018-01-01

Evolution of resistance to control measures in insect populations is a natural process, and management practices are intended to delay or mitigate resistance when it occurs. During the 2012/13 season the first reports of unexpected damage by Diatraea saccharalis on some Bt maize hybrids occurred in the northeast of San Luis province, Argentina. The affected Bt technologies were Herculex I® (HX-TC1507) and VT3PRO® (MON 89034 × MON 88017 * ). Event TC1507 expresses Cry1F and event MON 89034 expresses Cry1A.105 and Cry2Ab2, whichr are all Bt proteins with activity against the lepidopterans D. saccharalis and Spodoptera frugiperda (MON 88017 expresses the protein Cry3Bb1 for control of coleopteran insects and the enzyme CP4EPSPS for glyphosate tolerance). The affected area is an isolated region surrounded by sierra systems to the northeast and west, with a hot semi-arid climate, long frost-free period, warm winters, hot dry summers, and woody shrubs as native flora. To manage and mitigate the development of resistance, joint actions were taken by the industry, growers and Governmental Agencies. Hybrids expressing Vip3A protein (event MIR162) and/or Cry1Ab protein (events MON 810 and Bt11) as single or stacked events are used in early plantings to control the first generations of D. saccharalis , and in later plantings date's technologies with good control of S. frugiperda . A commitment was made to plant the refuge, and pest damage is monitored. As a result, maize production in the area is sustainable and profitable with yields above the average.

Management of Field-Evolved Resistance to Bt Maize in Argentina: A Multi-Institutional Approach

PubMed Central

Signorini, Ana M.; Abratti, Gustavo; Grimi, Damián; Machado, Marcos; Bunge, Florencia F.; Parody, Betiana; Ramos, Laura; Cortese, Pablo; Vesprini, Facundo; Whelan, Agustina; Araujo, Mónica P.; Podworny, Mariano; Cadile, Alejandro; Malacarne, María F.

2018-01-01

Evolution of resistance to control measures in insect populations is a natural process, and management practices are intended to delay or mitigate resistance when it occurs. During the 2012/13 season the first reports of unexpected damage by Diatraea saccharalis on some Bt maize hybrids occurred in the northeast of San Luis province, Argentina. The affected Bt technologies were Herculex I® (HX-TC1507) and VT3PRO® (MON 89034 × MON 88017*). Event TC1507 expresses Cry1F and event MON 89034 expresses Cry1A.105 and Cry2Ab2, whichr are all Bt proteins with activity against the lepidopterans D. saccharalis and Spodoptera frugiperda (MON 88017 expresses the protein Cry3Bb1 for control of coleopteran insects and the enzyme CP4EPSPS for glyphosate tolerance). The affected area is an isolated region surrounded by sierra systems to the northeast and west, with a hot semi-arid climate, long frost-free period, warm winters, hot dry summers, and woody shrubs as native flora. To manage and mitigate the development of resistance, joint actions were taken by the industry, growers and Governmental Agencies. Hybrids expressing Vip3A protein (event MIR162) and/or Cry1Ab protein (events MON 810 and Bt11) as single or stacked events are used in early plantings to control the first generations of D. saccharalis, and in later plantings date's technologies with good control of S. frugiperda. A commitment was made to plant the refuge, and pest damage is monitored. As a result, maize production in the area is sustainable and profitable with yields above the average. PMID:29888224

Use of a pollen-based diet to expose the ladybird beetle Propylea japonica to insecticidal proteins.

PubMed

Zhang, Xiaojie; Li, Yunhe; Romeis, Jörg; Yin, Xinming; Wu, Kongming; Peng, Yufa

2014-01-01

A rape seed pollen-based diet was developed and found to be suitable for use in a dietary exposure assay for Propylea japonica. Using the diet, we established and validated a dietary exposure assay by using the protease inhibitor E-64 as positive control. Dose-dependent responses were documented for all observed life-table parameters of P. japonica including survival, pupation and eclosion rates, development time and adult weight. Results suggested that the dietary assay can detect the effects of insecticidal compounds on the survival and development of P. japonica. Using the established dietary assay, we subsequently tested the toxicity of Cry1Ab, Cry1Ac and Cry1F proteins that are expressed by transgenic maize, cotton or rice plants to P. japonica larvae. The diet containing E-64 was included as a positive control. Survival and development of P. japonica larvae were not adversely affected when the diet contained purified Cry1Ab, Cry1Ac, or Cry1F at 500 µg/g diet representing a worst-case exposure scenario. In contrast, P. japonica larvae were adversely affected when the diet contained E-64. The bioactivity and stability of the Cry proteins in the diet and Cry protein uptake by the ladybird larvae were confirmed by bioassay with a Cry-sensitive insect species and by ELISA. The current study describes a suitable experimental system for assessing the potential effects of gut-active insecticidal compounds on ladybird beetle larvae. The experiments with the Cry proteins demonstrate that P. japonica larvae are not sensitive to Cry1Ab, Cry1Ac and Cry1F.

A Comprehensive Assessment of the Effects of Transgenic Cry1Ac/Cry1Ab Rice Huahui 1 on Adult Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae)

PubMed Central

Zhou, Xia; Guo, Yunling; Kong, Hua; Zuo, Jiao; Huang, Qixing; Jia, Ruizong; Guo, Anping; Xu, Lin

2016-01-01

Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae) is a widely distributed coleoptera predator in southern Asia in rice ecosystem, and adult M. discolor feed on both rice pollen and soft-bodied arthropods. Bitrophic bioassay and tritrophic bioassay were conducted to evaluate the potential impact of Cry1Ac/Cry1Ab-expressing rice Huahui 1 and its non-transgenic counterpart Minghui 63 on fitness parameters of adult M. discolor. The results showed that the survival, and fecundity of this beetle’ adults were not different when they fed on Bt rice or non-Bt rice pollen or Nilaparvata lugens (Stål) reared on Bt rice or non-Bt rice. Toxicity assessment to ensure M. discolor adults were not sensitive to Cry1Ab or Cry1Ac protein independent from the pollen background, M. discolor adults were fed with an artificial diet containing Cry1Ac, Cry1Ab or both protein approximately 10 times higher concentration than in Huahui 1 rice pollen. No difference was detected for any of the life-table parameters tested between Cry protein-containing and pure diet. Artificial diet containing E-64 (N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide) was included as a positive control. In contrast, the pre-oviposition and fecundity of M. discolor were significantly adversely affected by feeding on E-64-containing diet. In both bioassays, the uptakes of Cry protein by adult M. discolor were tested by ELISA measurements. These results indicated that adults of M. discolor are not affected by Cry1Ab- or Cry1Ac-expressing rice pollen and are not sensitive to Cry protein at concentrations exceeding the levels in rice pollen in Huahui1. This suggests that M. discolor adults would not be harmed by Cry1Ac/Cry1Ab rice if Bt rice Huahui 1 were commercialized. PMID:26914608

No Interactions of Stacked Bt Maize with the Non-target Aphid Rhopalosiphum padi and the Spider Mite Tetranychus urticae.

PubMed

Shu, Yinghua; Romeis, Jörg; Meissle, Michael

2018-01-01

In the agroecosystem, genetically engineered plants producing insecticidal Cry proteins from Bacillus thuringiensis (Bt) interact with non-target herbivores and other elements of the food web. Stacked Bt crops expose herbivores to multiple Cry proteins simultaneously. In this study, the direct interactions between SmartStax ® Bt maize producing six different Cry proteins and two herbivores with different feeding modes were investigated. Feeding on leaves of Bt maize had no effects on development time, fecundity, or longevity of the aphid Rhopalosiphum padi (Hemiptera: Aphididae), and no effects on the egg hatching time, development time, sex ratio, fecundity, and survival of the spider mite Tetranychus urticae (Acari: Tetranychidae). The results thus confirm the lack of effects on those species reported previously for some of the individual Cry proteins. In the Bt maize leaves, herbivore infestation did not result in a consistent change of Cry protein concentrations. However, occasional statistical differences between infested and non-infested leaves were observed for some Cry proteins and experimental repetitions. Overall, the study provides evidence that the Cry proteins in stacked Bt maize do not interact with two common non-target herbivores.

Investigations of immunogenic, allergenic and adjuvant properties of Cry1Ab protein after intragastric exposure in a food allergy model in mice.

PubMed

Andreassen, Monica; Bøhn, Thomas; Wikmark, Odd-Gunnar; Bodin, Johanna; Traavik, Terje; Løvik, Martinus; Nygaard, Unni Cecilie

2016-05-04

In genetically modified (GM) crops there is a risk that the inserted genes may introduce new allergens and/or adjuvants into the food and feed chain. The MON810 maize, expressing the insecticidal Cry1Ab toxin, is grown in many countries worldwide. In animal models, intranasal and intraperitoneal immunisations with the purified Cry1Ab proteins have induced immune responses, and feeding trials with Cry1Ab-containing feed have revealed some altered immune responses. Previous investigations have primarily measured antibody responses to the protein, while investigations of clinical food allergy symptoms, or allergy promotion (adjuvant effect) associated with the Cry1Ab protein are largely missing. We aimed to investigate immunogenic, allergenic and adjuvant properties of purified Cry1Ab toxin (trypCry1Ab, i.e., trypsin activated Cry1Ab) in a mouse model of food allergy. Female C3H/HeJ mice were immunized by intragastric gavage of 10 μg purified, trypsin activated Cry1Ab toxin (trypCry1Ab) alone or together with the food allergen lupin. Cholera toxin was added as a positive control for adjuvant effect to break oral tolerance. Clinical symptoms (anaphylaxis) as well as humoral and cellular responses were assessed. In contrast to results from previous airway investigations, we observed no indication of immunogenic properties of trypCry1Ab protein after repeated intragastric exposures to one dose, with or without CT as adjuvant. Moreover, the results indicated that trypCry1Ab given by the intragastric route was not able to promote allergic responses or anaphylactic reactions against the co-administered allergen lupin at the given dose. The study suggests no immunogenic, allergenic or adjuvant capacity of the given dose of trypCry1Ab protein after intragastric exposure of prime aged mice.

Analysis of cross-resistance to Vip3 proteins in eight insect colonies, from four insect species, selected for resistance to Bacillus thuringiensis insecticidal proteins.

PubMed

Gomis-Cebolla, Joaquín; Wang, Yuequin; Quan, Yudong; He, Kanglai; Walsh, Tom; James, Bill; Downes, Sharon; Kain, Wendy; Wang, Ping; Leonard, Kathy; Morgan, Tom; Oppert, Brenda; Ferré, Juan

2018-05-16

Bacillus thuringiensis Vip3 proteins are synthesized and secreted during the vegetative growth phase. They are activated by gut proteases, recognize and bind to midgut receptors, form pores and lyse cells. We tested the susceptibility to Vip3Aa and Vip3Ca of Cry1A-, Cry2A-, Dipel- and Vip3-resistant insect colonies from different species to determine whether resistance to other insecticidal proteins confers cross-resistance to Vip3 proteins. As expected, the colonies resistant to Cry1A proteins, Dipel (Helicoverpa armigera, Trichoplusia ni, Ostrinia furnacalis and Plodia interpunctella) or Cry2Ab (H. armigera and T. ni) were not cross-resistant to Vip3 proteins. In contrast, H. armigera colonies resistant to Vip3Aa or Vip3Aa/Cry2Ab showed cross-resistance to the Vip3Ca protein. Moreover, the Vip3Ca protein was highly toxic to O. furnacalis (LC 50 not significantly different from that of Cry1Ab), whereas the Vip3Aa protein only showed moderate growth inhibition at the highest concentration tested (100 µg/g of diet). These results extend the cross-resistance studies between Vip3 and Cry proteins, show for the first time cross-resistance between proteins within the Vip3 subfamily, and points to O. furnacalis as a target for the Vip3Ca protein. Copyright © 2018 Elsevier Inc. All rights reserved.

Lack of Detection of Bt Sugarcane Cry1Ab and NptII DNA and Proteins in Sugarcane Processing Products Including Raw Sugar

PubMed Central

Cheavegatti-Gianotto, Adriana; Gentile, Agustina; Oldemburgo, Danielle Angeloni; Merheb, Graciela do Amaral; Sereno, Maria Lorena; Lirette, Ron Peter; Ferreira, Thais Helena Silva; de Oliveira, Wladecir Salles

2018-01-01

Brazil is the largest sugarcane producer and the main sugar exporter in the world. The industrial processes applied by Brazilian mills are very efficient in producing highly purified sugar and ethanol. Literature presents evidence of lack of DNA/protein in these products, regardless of the nature of sugarcane used as raw material. Recently CTNBio, the Brazilian biosafety authority, has approved the first biotechnology-derived sugarcane variety for cultivation, event CTC175-A, which expresses the Cry1Ab protein to control the sugarcane borer (Diatraea saccharalis). The event also expresses neomycin-phosphotransferase type II (NptII) protein used as selectable marker during the transformation process. Because of the high purity of sugar and ethanol produced from genetically modified sugarcane, these end-products should potentially be classified as “pure substances, chemically defined,” by Brazilian Biosafety Law No. 11.105. If this classification is to be adopted, these substances are not considered as “GMO derivatives” and fall out of the scope of Law No. 11.105. In order to assess sugar composition and quality, we evaluate Cry1Ab and NptII expression in several sugarcane tissues and in several fractions from laboratory-scale processing of event CTC175-A for the presence of these heterologous proteins as well as for the presence of traces of recombinant DNA. The results of these studies show that CTC175-A presents high expression of Cry1Ab in leaves and barely detectable expression of heterologous proteins in stalks. We also evaluated the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase protein and DNA in the fractions of the industrial processing of conventional Brazilian sugarcane cultivars. Results from both laboratory and industrial processing were concordant, demonstrating that DNA and protein are not detected in the clarified juice and downstream processed fractions, including ethanol and raw sugar, indicating that protein and DNA are removed and/or degraded during processing. In conclusion, the processing of conventional sugarcane and CTC175-A Bt event results in downstream products with no detectable concentrations of heterologous DNA or new protein. These results help in the classification of sugar and ethanol derived from CTC175-A event as pure, chemically defined substances in Brazil and may relieve regulatory burdens in countries that import Brazilian sugar. PMID:29637070

Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa

PubMed Central

Chen, Lin; Jiang, Huang; Cheng, Qipeng; Chen, Junpeng; Wu, Gaobing; Kumar, Ashok; Sun, Ming; Liu, Ziduo

2015-01-01

Nematodes are known to be harmful to various crops, vegetables, plants and insects. The present study reports that, chitin upregulates the activity of chitinase (20%) and nematicidal potential (15%) of Pseudomonas aeruginosa. The chitinase gene (pachi) from P. aeruginosa was cloned, and its nematicidal activity of pachi protein against Caenorhabditis elegans was studied. The mortality rate induced by pachi increased by 6.3-fold when in association with Cry21Aa from Bacillus thuringiensis. Pachi efficiently killed C. elegans in its native state (LC50 = 387.3 ± 31.7 μg/ml), as well as in association with Cry21Aa (LC50 = 30.9 ± 4.1 μg/ml), by degrading the cuticle, egg shell and intestine in a relatively short time period of 24 h. To explore the nematidal potential of chitinase, six fusion proteins were constructed using gene engineering techniques. The CHACry showed higher activity against C. elegans than others owing to its high solubility. Notably, the CHACry showed a synergistic factor of 4.1 versus 3.5 a mixture [1:1] of pachi and Cry21Aa. The present study has identified eco-friendly biological routes (e.g., mixed proteins, fusion proteins) with potent nematicidal activity, which not only can help to prevent major crop losses but also strengthen the agro-economy and increase gross crop yield. PMID:26400097

Baseline sensitivity of maize borers in India to the Bacillus thuringiensis insecticidal proteins Cry1A.105 and Cry2Ab2.

PubMed

Jalali, Sushil K; Yadavalli, Lalitha; Ojha, Rakshit; Kumar, Pradyumn; Sulaikhabeevi, Suby B; Sharma, Reema; Nair, Rupa; Kadanur, Ravi C; Kamath, Subray P; Komarlingam, Mohan S

2015-08-01

Among the major pests of maize in India are two stem borers, Chilo partellus (Swinhoe) and Sesamia inferens (Walker), and an earworm, Helicoverpa armigera (Hübner). As a pest control strategy, transgenic Bacillus thuringiensis (Bt) maize hybrids are undergoing regulatory trials in India. We have determined the sensitivity of the target lepidopterans to the insecticidal Bt proteins expressed in Bt maize, as this determines product efficacy and the resistance management strategy to be adopted. Maize hybrids with event MON89034 express two insecticidal Bt proteins, Cry1A.105 and Cry2Ab2. Sensitivity profiles of 53 populations of C. partellus, 21 populations of S. inferens and 21 populations of H. armigera, collected between 2008 and 2013 from maize-growing areas in India, to Cry1A.105 and Cry2Ab2 proteins were generated through dose-response assays. Cry1A.105 protein was the most effective to neonates of C. partellus (mean MIC90 range 0.30-1.0 µg mL(-1) ) and H. armigera (mean MIC90 range 0.71-8.22 µg mL(-1) ), whereas Cry2Ab2 (mean MIC90 range 0.65-1.70 µg mL(-1) ) was the most effective to S. inferens. Populations of C. partellus, S. inferens and H. armigera were susceptible to the Bt proteins Cry1A.105 and Cry2Ab2. The Bt sensitivity data will serve as precommercialisation benchmarks for resistance monitoring purposes. © 2014 Society of Chemical Industry.

The interaction of two-spotted spider mites, Tetranychus urticae Koch, with Cry protein production and predation by Amblyseius andersoni (Chant) in Cry1Ac/Cry2Ab cotton and Cry1F maize.

USDA-ARS?s Scientific Manuscript database

Crops producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), are an important tool for managing lepidopteran pests on cotton and maize. However, the effects of these Bt crops on non-target organisms, especially natural enemies that provide biological control s...

The presence of Bacillus thuringiensis (Bt) protein in earthworms Eisenia fetida has no deleterious effects on their growth and reproduction.

PubMed

Shu, Yinghua; Ma, Honghui; Du, Yan; Li, Zhixian; Feng, Yuanjiao; Wang, Jianwu

2011-11-01

Earthworms Eisenia fetida, bred in substances with stover of two genetically-engineered Bacillus thuringiensis (Bt) corns (5422Bt1 (Event Bt11) and 5422CBCL (MON810)) expressing Cry1Ab and their near-isogenic non-Bt corn (5422), were used to investigate the non-target effects of Bt corn on soil-dwelling organisms. Cry1Ab concentrations in substances, casts and guts of E. fetida were also investigated by Enzyme-linked immunosorbent assay (ELISA). More than 90% individuals of E. fetida survived over a period of 30 d, irrespective of whether they received Bt corn or non-Bt corn. Compared to 5422 treatments, significantly higher relative growth rate and more number of new offspring and cocoons of E. fetida were found in 5422Bt1 and 5422CBCL treatments. These results were unlikely to be directly caused by Cry1Ab released from Bt corns but rather by differences in other factors of plants such as plant components (soluble sugar, total organic carbon, total protein and available phosphorus of Bt corns were more than 5422). ELISA results indicated immunoreactive Cry1Ab was detectable in substances, and the casts, guts of E. fetida from Bt corns treatments, of which the highest levels were detected in substances under the corresponding experimental conditions. With the increase of treated time, a strong decline was observed in Cry1Ab from substances and casts of E. fetida, whereas Cry1Ab in guts of E. fetida from 5422Bt1 treatments gradually increased and that from 5422CBCL treatments increased between 14 and 30 d. Therefore, the presence of Cry1Ab in E. fetida had no deleterious effects on their growth and reproduction. Copyright © 2011 Elsevier Ltd. All rights reserved.

Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cry1F Toxin Single-Chain Antibodies.

PubMed

Xu, Chongxin; Zhang, Cunzheng; Zhong, Jianfeng; Hu, Hui; Luo, Shimin; Liu, Xiaoqin; Zhang, Xiao; Liu, Yuan; Liu, Xianjin

2017-07-26

In the present study, a Cry1F-immunized rabbit phage display library (6.96 × 10 8 cfu/mL) was constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (a single-chain variable fragment, scFv) by biopanning. A total of 16 positive monoclonal phage scFv's were obtained after 4 rounds of panning, which were identified by enzyme-linked immunosorbent assay (ELISA), polymerized chain reaction, and DNA sequencing. The most positive phage scFv (named RF4) was expressed in Escherichia coli HB2151, and a soluble protein of approximately 30 kDa was purified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An indirect competitive ELISA (IC-ELISA) was established on the basis of purified soluble RF4-scFv for Cry1F toxin. It indicated the 50% inhibition of the control (IC 50 ) was 11.56 ng/mL and the detection limit (IC 10 ) was 0.18 ng/mL and showed weak cross-reactivities for Cry1Ab (2.8%), Cry1Ac (1.3%), and Cry1B, Cry1C, Cry1Ie, and Cry2A (less than 0.1%). It was found that IC-ELISA detected Cry1F toxin spiked in rice, wheat, corn, and soil samples with good accuracy, stability, and repeatability. The recoveries were in the range of 80.2-99.6%, and the coefficients of variation were in the range of 2.5-10.0%. These results showed that IC-ELISA based on scFv from the immunized rabbit phage display library was promising for specific detection of Cry1F toxin in agroproducts and environmental samples.

The blue light-induced interaction of cryptochrome 1 with COP1 requires SPA proteins during Arabidopsis light signaling.

PubMed

Holtkotte, Xu; Ponnu, Jathish; Ahmad, Margaret; Hoecker, Ute

2017-10-01

Plants constantly adjust their growth, development and metabolism to the ambient light environment. Blue light is sensed by the Arabidopsis photoreceptors CRY1 and CRY2 which subsequently initiate light signal transduction by repressing the COP1/SPA E3 ubiquitin ligase. While the interaction between cryptochromes and SPA is blue light-dependent, it was proposed that CRY1 interacts with COP1 constitutively, i.e. also in darkness. Here, our in vivo co-immunoprecipitation experiments suggest that CRY1 and CRY2 form a complex with COP1 only after seedlings were exposed to blue light. No association between COP1 and CRY1 or CRY2 was observed in dark-grown seedlings. Thus, our results suggest that cryptochromes bind the COP1/SPA complex after photoactivation by blue light. In a spa quadruple mutant that is devoid of all four SPA proteins, CRY1 and COP1 did not interact in vivo, neither in dark-grown nor in blue light-grown seedlings. Hence, SPA proteins are required for the high-affinity interaction between CRY1 and COP1 in blue light. Yeast three-hybrid experiments also show that SPA1 enhances the CRY1-COP1 interaction. The coiled-coil domain of SPA1 which is responsible for COP1-binding was necessary to mediate a CRY1-SPA1 interaction in vivo, implying that-in turn-COP1 may be necessary for a CRY1-SPA1 complex formation. Hence, SPA1 and COP1 may act cooperatively in recognizing and binding photoactivated CRY1. In contrast, the blue light-induced association between CRY2 and COP1 was not dependent on SPA proteins in vivo. Similarly, ΔCC-SPA1 interacted with CRY2, though with a much lower affinity than wild-type SPA1. In total, our results demonstrate that CRY1 and CRY2 strongly differ in their blue light-induced interaction with the COP1/SPA complex.

Genomic sequences of murine gamma B- and gamma C-crystallin-encoding genes: promoter analysis and complete evolutionary pattern of mouse, rat and human gamma-crystallins.

PubMed

Graw, J; Liebstein, A; Pietrowski, D; Schmitt-John, T; Werner, T

1993-12-22

The murine genes, gamma B-cry and gamma C-cry, encoding the gamma B- and gamma C-crystallins, were isolated from a genomic DNA library. The complete nucleotide (nt) sequences of both genes were determined from 661 and 711 bp, respectively, upstream from the first exon to the corresponding polyadenylation sites, comprising more than 2650 and 2890 bp, respectively. The new sequences were compared to the partial cDNA sequences available for the murine gamma B-cry and gamma C-cry, as well as to the corresponding genomic sequences from rat and man, at both the nt and predicted amino acid (aa) sequence levels. In the gamma B-cry promoter region, a canonical CCAAT-box, a TATA-box, putative NF-I and C/EBP sites were detected. An R-repeat is inserted 366 bp upstream from the transcription start point. In contrast, the gamma C-cry promoter does not contain a CCAAT-box, but some other putative binding sites for transcription factors (AP-2, UBP-1, LBP-1) were located by computer analysis. The promoter regions of all six gamma-cry from mouse, rat and human, except human psi gamma F-cry, were analyzed for common sequence elements. A complex sequence element of about 70-80 bp was found in the proximal promoter, which contains a gamma-cry-specific and almost invariant sequence (crygpel) of 14 nt, and ends with the also invariant TATA-box. Within the complex sequence element, a minimum of three further features specific for the gamma A-, gamma B- and gamma D/E/F-cry genes can be defined, at least two of which were recently shown to be functional. In addition to these four sequence elements, a subtype-specific structure of inverted repeats with different-sized spacers can be deduced from the multiple sequence alignment. A phylogenetic analysis based on the promoter region, as well as the complete exon 3 of all gamma-cry from mouse, rat and man, suggests separation of only five gamma-cry subtypes (gamma A-, gamma B-, gamma C-, gamma D- and gamma E/F-cry) prior to species separation.

Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis.

PubMed

Martins, Erica Soares; Monnerat, Rose Gomes; Queiroz, Paulo Roberto; Dumas, Vinicius Fiuza; Braz, Shélida Vasconcelos; de Souza Aguiar, Raimundo Wagner; Gomes, Ana Cristina Menezes Mendes; Sánchez, Jorge; Bravo, Alejandra; Ribeiro, Bergmann Morais

2010-02-01

Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis. 2009. Published by Elsevier Ltd.

Molecular studies of segmental aneusomy: FISHing for the atypical cry in del(5)(p15.3).

PubMed

Hodge, J C; Lawson-Yuen, A; Stoler, J M; Ligon, A H

2007-01-01

We report a newborn male with multiple congenital anomalies including growth retardation, hypotonia, dysmorphic facies, widely-spaced nipples, micropenis, cryptorchidism, optic nerve hypoplasia, heart disease, and a striking, high-pitched cry. Chromosome analysis revealed de novo partial trisomy 11q due to a der(5)t(5;11)(p15.3;q22). Fluorescence in situ hybridization (FISH) showed loss of the 5p telomere signal on the der(5) chromosome, indicating the infant has partial monosomy 5p in addition to partial trisomy 11q. Among cases involving trisomy 11q, an unusual cry has only been documented in the presence of a der(5)t(5p;11q). This apparent dependence of the abnormal cry on monosomy 5p suggested the same genetic mechanism that occurs in Cri du chat syndrome (CDCS) may be responsible for the atypical cry in der(5)t(5p;11q) individuals. Neither a commercial CDCS probe (LSI D5S23, D5S721) nor a series of BAC clones encompassing distal regions implicated in the CDCS-associated cat-cry were deleted in our patient. These results suggest a second cry-modifying locus maps telomeric to BAC RP11-94J21 in band 5p15.33. This locus may not only cause the abnormal cry in individuals with a der(5)t(5p;11q) but could also contribute to the phenotypic variability and discordant mapping studies observed for CDCS. Copyright (c) 2007 S. Karger AG, Basel.

Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

PubMed

Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

2016-09-01

The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. Copyright © 2016 Elsevier B.V. All rights reserved.

The fate of fusion Cry1Ab/1Ac proteins from Bt-transgenic rice in soil and water.

PubMed

Liu, Yongbo; Li, Junsheng; Luo, Zunlan; Wang, Huaru; Liu, Fang

2016-02-01

Toxin proteins form transgenic crops entering into the environment are likely affect non-target organisms. To investigate the entry route and fate of fusion Cry1Ab/1Ac proteins from transgenic rice expressing insecticide toxins from Bacillus thuringiensis (Bt) in soil and water, we conducted greenhouse and field experiments in 2013 and 2014. Cry1Ab/1Ac proteins from Bt-transgenic rice in soil was found within a horizontal range of 25cm, where most of plant roots distributed. Concentration of Cry1Ab/1Ac proteins was lower in water than in soil in the greenhouse experiment, and no Cry1Ab/1Ac protein was detected in field water. Cry1Ab/1Ac concentration from rice straws was higher in ditch water than in distilled water due to the existence of aquatic organisms in ditch water. Bt proteins from transgenic crops enter into soil ecosystems mainly through root exudates and into aquatic ecosystems through plant residues, which determines Bt fate in the environment. Copyright © 2015 Elsevier Inc. All rights reserved.

Consumption of Bt Maize Pollen Containing Cry1Ie Does Not Negatively Affect Propylea japonica (Thunberg) (Coleoptera: Coccinellidae).

PubMed

Li, Yonghui; Liu, Yanmin; Yin, Xinming; Romeis, Jörg; Song, Xinyuan; Chen, Xiuping; Geng, Lili; Peng, Yufa; Li, Yunhe

2017-03-16

Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) are prevalent predators and pollen feeders in East Asian maize fields. They are therefore indirectly (via prey) and directly (via pollen) exposed to Cry proteins within Bt -transgenic maize fields. The effects of Cry1Ie-producing transgenic maize pollen on the fitness of P. japonica was assessed using two dietary-exposure experiments in the laboratory. In the first experiment, survival, larval developmental time, adult fresh weight, and fecundity did not differ between ladybirds consuming Bt or non- Bt maize pollen. In the second experiment, none of the tested lethal and sublethal parameters of P. japonica were negatively affected when fed a rapeseed pollen-based diet containing Cry1Ie protein at 200 μg/g dry weight of diet. In contrast, the larval developmental time, adult fresh weight, and fecundity of P. japonica were significantly adversely affected when fed diet containing the positive control compound E-64. In both experiments, the bioactivity of the Cry1Ie protein in the food sources was confirmed by bioassays with a Cry1Ie-sensitive lepidopteran species. These results indicated that P. japonica are not affected by the consumption of Cry1Ie-expressing maize pollen and are not sensitive to the Cry1Ie protein, suggesting that the growing of Bt maize expressing Cry1Ie protein will pose a negligible risk to P. japonica .

Evaluation of sampling plans to detect Cry9C protein in corn flour and meal.

PubMed

Whitaker, Thomas B; Trucksess, Mary W; Giesbrecht, Francis G; Slate, Andrew B; Thomas, Francis S

2004-01-01

StarLink is a genetically modified corn that produces an insecticidal protein, Cry9C. Studies were conducted to determine the variability and Cry9C distribution among sample test results when Cry9C protein was estimated in a bulk lot of corn flour and meal. Emphasis was placed on measuring sampling and analytical variances associated with each step of the test procedure used to measure Cry9C in corn flour and meal. Two commercially available enzyme-linked immunosorbent assay kits were used: one for the determination of Cry9C protein concentration and the other for % StarLink seed. The sampling and analytical variances associated with each step of the Cry9C test procedures were determined for flour and meal. Variances were found to be functions of Cry9C concentration, and regression equations were developed to describe the relationships. Because of the larger particle size, sampling variability associated with cornmeal was about double that for corn flour. For cornmeal, the sampling variance accounted for 92.6% of the total testing variability. The observed sampling and analytical distributions were compared with the Normal distribution. In almost all comparisons, the null hypothesis that the Cry9C protein values were sampled from a Normal distribution could not be rejected at 95% confidence limits. The Normal distribution and the variance estimates were used to evaluate the performance of several Cry9C protein sampling plans for corn flour and meal. Operating characteristic curves were developed and used to demonstrate the effect of increasing sample size on reducing false positives (seller's risk) and false negatives (buyer's risk).

Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons.

PubMed

Bolte, Petra; Bleibaum, Florian; Einwich, Angelika; Günther, Anja; Liedvogel, Miriam; Heyers, Dominik; Depping, Anne; Wöhlbrand, Lars; Rabus, Ralf; Janssen-Bienhold, Ulrike; Mouritsen, Henrik

2016-01-01

Cryptochromes are ubiquitously expressed in various animal tissues including the retina. Some cryptochromes are involved in regulating circadian activity. Cryptochrome proteins have also been suggested to mediate the primary mechanism in light-dependent magnetic compass orientation in birds. Cryptochrome 1b (Cry1b) exhibits a unique carboxy terminus exclusively found in birds so far, which might be indicative for a specialised function. Cryptochrome 1a (Cry1a) is so far the only cryptochrome protein that has been localised to specific cell types within the retina of migratory birds. Here we show that Cry1b, an alternative splice variant of Cry1a, is also expressed in the retina of migratory birds, but it is primarily located in other cell types than Cry1a. This could suggest different functions for the two splice products. Using diagnostic bird-specific antibodies (that allow for a precise discrimination between both proteins), we show that Cry1b protein is found in the retinae of migratory European robins (Erithacus rubecula), migratory Northern Wheatears (Oenanthe oenanthe) and pigeons (Columba livia). In all three species, retinal Cry1b is localised in cell types which have been discussed as potentially well suited locations for magnetoreception: Cry1b is observed in the cytosol of ganglion cells, displaced ganglion cells, and in photoreceptor inner segments. The cytosolic rather than nucleic location of Cry1b in the retina reported here speaks against a circadian clock regulatory function of Cry1b and it allows for the possible involvement of Cry1b in a radical-pair-based magnetoreception mechanism.

Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a computational pipeline.

PubMed

Ye, Weixing; Zhu, Lei; Liu, Yingying; Crickmore, Neil; Peng, Donghai; Ruan, Lifang; Sun, Ming

2012-07-01

We have designed a high-throughput system for the identification of novel crystal protein genes (cry) from Bacillus thuringiensis strains. The system was developed with two goals: (i) to acquire the mixed plasmid-enriched genomic sequence of B. thuringiensis using next-generation sequencing biotechnology, and (ii) to identify cry genes with a computational pipeline (using BtToxin_scanner). In our pipeline method, we employed three different kinds of well-developed prediction methods, BLAST, hidden Markov model (HMM), and support vector machine (SVM), to predict the presence of Cry toxin genes. The pipeline proved to be fast (average speed, 1.02 Mb/min for proteins and open reading frames [ORFs] and 1.80 Mb/min for nucleotide sequences), sensitive (it detected 40% more protein toxin genes than a keyword extraction method using genomic sequences downloaded from GenBank), and highly specific. Twenty-one strains from our laboratory's collection were selected based on their plasmid pattern and/or crystal morphology. The plasmid-enriched genomic DNA was extracted from these strains and mixed for Illumina sequencing. The sequencing data were de novo assembled, and a total of 113 candidate cry sequences were identified using the computational pipeline. Twenty-seven candidate sequences were selected on the basis of their low level of sequence identity to known cry genes, and eight full-length genes were obtained with PCR. Finally, three new cry-type genes (primary ranks) and five cry holotypes, which were designated cry8Ac1, cry7Ha1, cry21Ca1, cry32Fa1, and cry21Da1 by the B. thuringiensis Toxin Nomenclature Committee, were identified. The system described here is both efficient and cost-effective and can greatly accelerate the discovery of novel cry genes.

Ingestion of Bt corn pollen containing Cry1Ab/2Aj or Cry1Ac does not harm Propylea japonica larvae

PubMed Central

Liu, Yanmin; Liu, Qingsong; Wang, Yanan; Chen, Xiuping; Song, Xinyuan; Romeis, Jörg; Li, Yunhe; Peng, Yufa

2016-01-01

Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) is a prevalent pollen consumer in corn fields and is therefore exposed to insecticidal proteins contained in the pollen of insect-resistant transgenic corn cultivars expressing Cry proteins derived from Bacillus thuringiensis (Bt). In the present study, the potential effect of Cry1Ab/2Aj- or Cry1Ac-containing transgenic Bt corn pollen on the fitness of P. japonica larvae was evaluated. The results show that the larval developmental time was significantly shorter when P. japonica larvae were fed pollen from Bt corn cultivars rather than control pollen but that pupation rate, eclosion rate, and adult fresh weight were not significantly affected. In the feeding experiments, the stability of the Cry proteins in the food sources was confirmed. When Bt corn pollen passed through the gut of P. japonica, 23% of Cry1Ab/2Aj was digested. The results demonstrate that consumption of Bt corn pollen containing Cry1Ab/2Aj or Cry1Ac has no detrimental effect on P. japonica larvae; the shortened developmental time of larvae that consumed these proteins was likely attributable to unknown differences in the nutritional composition between the Bt-transgenic and control corn pollen. PMID:27005950

Aberrant rhythmic expression of cryptochrome2 regulates the radiosensitivity of rat gliomas.

PubMed

Fan, Wang; Caiyan, Li; Ling, Zhu; Jiayun, Zhao

2017-09-29

In this study, we investigated the role of the clock regulatory protein cryptochrome 2 (Cry2) in determining the radiosensitivity of C6 glioma cells in a rat model. We observed that Cry2 mRNA and protein levels showed aberrant rhythmic periodicity of 8 h in glioma tissues, compared to 24 h in normal brain tissue. Cry2 mRNA and protein levels did not respond to irradiation in normal tissues, but both were increased at the ZT4 (low Cry2) and ZT8 (high Cry2) time points in gliomas. Immunohistochemical staining of PCNA and TUNEL assays demonstrated that high Cry2 expression in glioma tissues was associated with increased cell proliferation and decreased apoptosis. Western blot analysis showed that glioma cell fate was independent of p53, but was probably dependent on p73, which was more highly expressed at ZT4 (low Cry2) than at ZT8 (high Cry2). Levels of both p53 and p73 were unaffected by irradiation in normal brain tissues. These findings suggest aberrant rhythmic expression of Cry2 influence on radiosensitivity in rat gliomas.

Molecular characterization of Cry1D-133 toxin from Bacillus thuringiensis strain HD133 and its toxicity against Spodoptera littoralis.

PubMed

BenFarhat-Touzri, Dalel; Driss, Fatma; Jemli, Sonia; Tounsi, Slim

2018-06-01

Bacillus thuringiensis subsp. aizawai strain HD133, known by its effectiveness against Spodoptera species, produces bipyramidal crystals encompassing the insecticidal proteins Cry1Ab, Cry1Ca and Cry1D-133 in the proportions 60:37:3, respectively. In this study, we dealt with the relevance of the low rate of Cry1D-133. The cry1D-133 gene from HD133 was cloned and sequenced. Both nucleotide and amino acid sequence similarity analyses with the cry1D genes available in the GenBank database revealed that cry1D-133 is a new variant of cry1Da-type genes with 99% identity with cry1Da1. Molecular modeling of the Cry1D-133 toxin showed that its higher toxicity is correlated to a higher number of toxin-receptor interactions. Optimal culture conditions of 4 h post-induction time, 160 rpm agitation and 37 °C post-induction temperature were determined and adopted to overproduce Cry1D-133 toxin at adequate amounts to carryout bioassays. A gradual increase of the proportion of Cry1D-133 to the HD133 insecticidal proteins forming the crystal (Cry1D-133, Cry1Ca and Cry1Ab) showed an improvement of the toxicity against Spodoptera littoralis larvae. Therefore, the potential of Cry1D-133 to enhance HD133 toxicity could promote its combination with other B. thuringiensis insecticidal proteins toxins in order to increase target range or to delay the emergence of resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

Sodium Solute Symporter and Cadherin Proteins Act as Bacillus thuringiensis Cry3Ba Toxin Functional Receptors in Tribolium castaneum*

PubMed Central

Contreras, Estefanía; Schoppmeier, Michael; Real, M. Dolores; Rausell, Carolina

2013-01-01

Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins. In contrast, TcAPN-I silencing had no effect on Cry3Ba larval toxicity, suggesting that this protein is not relevant in the Cry3Ba toxin mode of action in Tc. Remarkable features of TcSSS protein were the presence of cadherin repeats in its amino acid sequence and that a TcSSS peptide fragment containing a sequence homologous to a binding epitope found in Manduca sexta and Tenebrio molitor Bt cadherin functional receptors enhanced Cry3Ba toxicity. This is the first time that the involvement of a sodium solute symporter protein as a Bt functional receptor has been demonstrated. The role of this novel receptor in Bt toxicity against coleopteran insects together with the lack of receptor functionality of aminopeptidase N proteins might account for some of the differences in toxin specificity between Lepidoptera and Coleoptera insect orders. PMID:23645668

Activity of Bacillus thuringiensis Cry1Ie2, Cry2Ac7, and Cry7Ab3 proteins against Anticarsia gemmatalis, Chrysodeixis includens and Ceratoma trifurcata

USDA-ARS?s Scientific Manuscript database

Transgenic soybeans producing the Cry1Ac insecticidal protein from the bacterium Bacillus thuringiensis (or “Bt”) are currently used to control larvae of the velvetbean caterpillar (Anticarsia gemmatalis Hübner) and the soybean looper [Chrysodeixis includens (Walker)]. The main threat to the sustain...

Development of Monoclonal Antibodies Recognizing Linear Epitope: Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton, Maize, and Tobacco.

PubMed

Cao, Zhen; Zhang, Wei; Ning, Xiangxue; Wang, Baomin; Liu, Yunjun; Li, Qing X

2017-11-22

Bacillus thuringiensis Cry1Ac, Cry1Ia1, and Cry1Ie are δ-endotoxin insecticidal proteins widely implemented in genetically modified organisms (GMO), such as cotton, maize, and potato. Western blot assay integrates electrophoresis separation power and antibody high specificity for monitoring specific exogenous proteins expressed in GMO. Procedures for evoking monoclonal antibody (mAb) for Western blot were poorly documented. In the present study, Cry1Ac partially denatured at 100 °C for 5 min was used as an immunogen to develop mAbs selectively recognizing a linear epitope of Cry1Ac for Western blot. mAb 5E9C6 and 3E6E2 selected with sandwich ELISA strongly recognized the heat semidenatured Cry1Ac. Particularly, 3E6E2 recognized both E. coli and cotton seed expressed Cry1Ac in Western blot. Such strategy of using partially denatured proteins as immunogens and using sandwich ELISA for mAb screening was also successfully demonstrated with production of mAbs against Cry1Ie for Western blot assay in maize.

Susceptibility of field populations of the fall armyworm (Lepidoptera: Noctuidae) from Florida and Puerto Rico to purified Cry1F protein and corn leaf tissue containing single and pyramided Bt genes

USDA-ARS?s Scientific Manuscript database

Larval survival of Cry1F-susceptible (FL), -resistant (PR and Cry1F-RR), and -heterozygous (FL x PR and Cry1F-RS) populations of the fall armyworm, Spodoptera frugiperda (J.E. Smith) to purified Cry1F protein and corn leaf tissue of seven Bacillus thuringiensis (Bt) corn hybrids and five non-Bt corn...

Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane

PubMed Central

2017-01-01

The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests. PMID:28123906

DNA barcoding simplifies environmental risk assessment of genetically modified crops in biodiverse regions.

PubMed

Nzeduru, Chinyere V; Ronca, Sandra; Wilkinson, Mike J

2012-01-01

Transgenes encoding for insecticidal crystal (Cry) proteins from the soil-dwelling bacterium Bacillus Thuringiensis have been widely introduced into Genetically Modified (GM) crops to confer protection against insect pests. Concern that these transgenes may also harm beneficial or otherwise valued insects (so-called Non Target Organisms, NTOs) represents a major element of the Environmental Risk Assessments (ERAs) used by all countries prior to commercial release. Compiling a comprehensive list of potentially susceptible NTOs is therefore a necessary part of an ERA for any Cry toxin-containing GM crop. In partly-characterised and biodiverse countries, NTO identification is slowed by the need for taxonomic expertise and time to enable morphological identifications. This limitation represents a potentially serious barrier to timely adoption of GM technology in some developing countries. We consider Bt Cry1A cowpea (Vigna unguiculata) in Nigeria as an exemplar to demonstrate how COI barcoding can provide a simple and cost-effective means of addressing this problem. Over a period of eight weeks, we collected 163 insects from cowpea flowers across the agroecological and geographic range of the crop in Nigeria. These individuals included 32 Operational Taxonomic Units (OTUs) spanning four Orders and that could mostly be assigned to genus or species level. They included 12 Lepidopterans and two Coleopterans (both potentially sensitive to different groups of Cry proteins). Thus, barcode-assisted diagnoses were highly harmonised across groups (typically to genus or species level) and so were insensitive to expertise or knowledge gaps. Decisively, the entire study was completed within four months at a cost of less than 10,000 US$. The broader implications of the findings for food security and the capacity for safe adoption of GM technology are briefly explored.

Laboratory toxicity studies demonstrate no adverse effects of Cry1Ab and Cry3Bb1 to larvae of Adalia bipunctata (Coleoptera: Coccinellidae): the importance of study design.

PubMed

Alvarez-Alfageme, Fernando; Bigler, Franz; Romeis, Jörg

2011-06-01

Scientific studies are frequently used to support policy decisions related to transgenic crops. Schmidt et al., Arch Environ Contam Toxicol 56:221-228 (2009) recently reported that Cry1Ab and Cry3Bb were toxic to larvae of Adalia bipunctata in direct feeding studies. This study was quoted, among others, to justify the ban of Bt maize (MON 810) in Germany. The study has subsequently been criticized because of methodological shortcomings that make it questionable whether the observed effects were due to direct toxicity of the two Cry proteins. We therefore conducted tritrophic studies assessing whether an effect of the two proteins on A. bipunctata could be detected under more realistic routes of exposure. Spider mites that had fed on Bt maize (events MON810 and MON88017) were used as carriers to expose young A. bipunctata larvae to high doses of biologically active Cry1Ab and Cry3Bb1. Ingestion of the two Cry proteins by A. bipunctata did not affect larval mortality, weight, or development time. These results were confirmed in a subsequent experiment in which A. bipunctata were directly fed with a sucrose solution containing dissolved purified proteins at concentrations approximately 10 times higher than measured in Bt maize-fed spider mites. Hence, our study does not provide any evidence that larvae of A. bipunctata are sensitive to Cry1Ab and Cry3Bb1 or that Bt maize expressing these proteins would adversely affect this predator. The results suggest that the apparent harmful effects of Cry1Ab and Cry3Bb1 reported by Schmidt et al., Arch Environ Contam Toxicol 56:221-228 (2009) were artifacts of poor study design and procedures. It is thus important that decision-makers evaluate the quality of individual scientific studies and do not view all as equally rigorous and relevant.

40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

Code of Federal Regulations, 2010 CFR

2010-07-01

... protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532... thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn...

40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

Code of Federal Regulations, 2011 CFR

2011-07-01

... protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532... thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn...

40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

Code of Federal Regulations, 2012 CFR

2012-07-01

... protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532... thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn...

40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... protein in corn; exemption from the requirement of a tolerance. 174.532 Section 174.532 Protection of...Cry3.1Ab protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn, field; corn...

40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... protein in corn; exemption from the requirement of a tolerance. 174.532 Section 174.532 Protection of...Cry3.1Ab protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn, field; corn...

A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy

PubMed Central

Zeng, Xianxu; Tate, Rebecca E.; McKee, Mary L.; Capen, Diane E.; Zhang, Zhan; Tanzi, Rudolph E.; Zhou, Chao

2015-01-01

Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function. PMID:26348211

Structure of Full-length Drosophila Cryptochrome

PubMed Central

Zoltowski, Brian D.; Vaidya, Anand T.; Top, Deniz; Widom, Joanne; Young, Michael W.; Crane, Brian R.

2011-01-01

The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions. PMID:22080955

Cyt1Aa protein of Bacillus thuringiensis is toxic to the Cottonwood Leaf Beetle, Chrysomela scripta, and suppresses high levels of resistance to Cry3Aa

Treesearch

Brian A. Federici; Leah S. Bauer

1998-01-01

The insecticidal activity of Bacillus thuringiensis is due primarily to Cry and Cyt proteins. Cry proteins are typically toxic to lepidopterous, coleopterous, or dipterous insects, whereas the known toxicity of Cyt proteins is limited to dipterans. We report here that a Cyt protein, Cyt1Aa, is also highly toxic to the cottonwood leaf beetle, Chrysomela scripta, with a...

40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed; cotton...

40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed; cotton...

40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a plant...

40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a plant...

40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a plant...

40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a...

Code of Federal Regulations, 2011 CFR

2011-07-01

... in cotton; temporary exemption from the requirement of a tolerance. 174.530 Section 174.530... thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food commodities of cotton, cotton; cotton, undelinted...

40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a plant...

40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a...

Code of Federal Regulations, 2010 CFR

2010-07-01

... in cotton; temporary exemption from the requirement of a tolerance. 174.530 Section 174.530... thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food commodities of cotton, cotton; cotton, undelinted...

40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed; cotton...

Cry1A(b)16 toxin from Bacillus thuringiensis: Theoretical refinement of three-dimensional structure and prediction of peptides as molecular markers for detection of genetically modified organisms.

PubMed

Plácido, Alexandra; Coelho, Andreia; Abreu Nascimento, Lucas; Gomes Vasconcelos, Andreanne; Fátima Barroso, Maria; Ramos-Jesus, Joilson; Costa, Vladimir; das Chagas Alves Lima, Francisco; Delerue-Matos, Cristina; Martins Ramos, Ricardo; Marani, Mariela M; Roberto de Souza de Almeida Leite, José

2017-07-01

Transgenic maize produced by the insertion of the Cry transgene into its genome became the second most cultivated crop worldwide. Cry gene from Bacillus thuringiensis kurstaki expresses protein derivatives of crystalline endotoxins which confer insect resistance onto the maize crop. Mandatory labeling of processed food containing or made by genetically modified organisms is in force in many countries, so, it is very urgent to develop fast and practical methods for GMO identification, for example, biosensors. In the absence of an available empirical structure of Cry1A(b)16 protein, a theoretical model was effectively generated, in this work, by homology modeling and molecular dynamics simulations based on two available homologous protein structures. Molecular dynamics simulations were carried out to refine the selected model, and an analysis of its global structure was performed. The refined models of Cry1A(b)16 showed a standard fold and structural characteristics similar to those seen in Bacillus thuringiensis Cry1A(a) insecticidal toxin and Bacillus thuringiensis serovar kurstaki Cry1A(c) toxin. After in silico analysis of Cry1A(b)16, two immunoreactive candidate peptides were selected and specific polyclonal antibodies were produced resulting in antibody-peptide interaction. Biosensing devices are expected to be developed for detection of the Cry1A(b) protein as a marker of transgenic maize in food. Proteins 2017; 85:1248-1257. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Effect of Stacked Insecticidal Cry Proteins from Maize Pollen on Nurse Bees (Apis mellifera carnica) and Their Gut Bacteria

PubMed Central

Härtel, Stephan; Näther, Astrid; Dohrmann, Anja B.; Steffan-Dewenter, Ingolf; Tebbe, Christoph C.

2013-01-01

Honey bee pollination is a key ecosystem service to nature and agriculture. However, biosafety research on genetically modified crops rarely considers effects on nurse bees from intact colonies, even though they receive and primarily process the largest amount of pollen. The objective of this study was to analyze the response of nurse bees and their gut bacteria to pollen from Bt maize expressing three different insecticidal Cry proteins (Cry1A.105, Cry2Ab2, and Cry3Bb1). Naturally Cry proteins are produced by bacteria (Bacillus thuringiensis). Colonies of Apis mellifera carnica were kept during anthesis in flight cages on field plots with the Bt maize, two different conventionally bred maize varieties, and without cages, 1-km outside of the experimental maize field to allow ad libitum foraging to mixed pollen sources. During their 10-days life span, the consumption of Bt maize pollen had no effect on their survival rate, body weight and rates of pollen digestion compared to the conventional maize varieties. As indicated by ELISA-quantification of Cry1A.105 and Cry3Bb1, more than 98% of the recombinant proteins were degraded. Bacterial population sizes in the gut were not affected by the genetic modification. Bt-maize, conventional varieties and mixed pollen sources selected for significantly different bacterial communities which were, however, composed of the same dominant members, including Proteobacteria in the midgut and Lactobacillus sp. and Bifidobacterium sp. in the hindgut. Surprisingly, Cry proteins from natural sources, most likely B. thuringiensis, were detected in bees with no exposure to Bt maize. The natural occurrence of Cry proteins and the lack of detectable effects on nurse bees and their gut bacteria give no indication for harmful effects of this Bt maize on nurse honey bees. PMID:23533634

Effect of stacked insecticidal Cry proteins from maize pollen on nurse bees (Apis mellifera carnica) and their gut bacteria.

PubMed

Hendriksma, Harmen P; Küting, Meike; Härtel, Stephan; Näther, Astrid; Dohrmann, Anja B; Steffan-Dewenter, Ingolf; Tebbe, Christoph C

2013-01-01

Honey bee pollination is a key ecosystem service to nature and agriculture. However, biosafety research on genetically modified crops rarely considers effects on nurse bees from intact colonies, even though they receive and primarily process the largest amount of pollen. The objective of this study was to analyze the response of nurse bees and their gut bacteria to pollen from Bt maize expressing three different insecticidal Cry proteins (Cry1A.105, Cry2Ab2, and Cry3Bb1). Naturally Cry proteins are produced by bacteria (Bacillus thuringiensis). Colonies of Apis mellifera carnica were kept during anthesis in flight cages on field plots with the Bt maize, two different conventionally bred maize varieties, and without cages, 1-km outside of the experimental maize field to allow ad libitum foraging to mixed pollen sources. During their 10-days life span, the consumption of Bt maize pollen had no effect on their survival rate, body weight and rates of pollen digestion compared to the conventional maize varieties. As indicated by ELISA-quantification of Cry1A.105 and Cry3Bb1, more than 98% of the recombinant proteins were degraded. Bacterial population sizes in the gut were not affected by the genetic modification. Bt-maize, conventional varieties and mixed pollen sources selected for significantly different bacterial communities which were, however, composed of the same dominant members, including Proteobacteria in the midgut and Lactobacillus sp. and Bifidobacterium sp. in the hindgut. Surprisingly, Cry proteins from natural sources, most likely B. thuringiensis, were detected in bees with no exposure to Bt maize. The natural occurrence of Cry proteins and the lack of detectable effects on nurse bees and their gut bacteria give no indication for harmful effects of this Bt maize on nurse honey bees.

Field Trial Performance of Herculex XTRA (Cry34Ab1/Cry35Ab1) and SmartStax (Cry34Ab1/Cry35Ab1 + Cry3Bb1) Hybrids and Soil Insecticides Against Western and Northern Corn Rootworms (Coleoptera: Chrysomelidae).

PubMed

Johnson, K D; Campbell, L A; Lepping, M D; Rule, D M

2017-06-01

Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), and northern corn rootworm, Diabrotica barberi Smith and Lawrence (Coleoptera: Chrysomelidae), are important insect pests in corn, Zea mays L. For more than a decade, growers have been using transgenic plants expressing proteins from the bacterium Bacillus thuringiensis (Bt) to protect corn roots from feeding. In 2011, western corn rootworm populations were reported to have developed resistance to Bt hybrids expressing Cry3Bb1 and later found to be cross-resistant to hybrids expressing mCry3A and eCry3.1Ab. The identification of resistance to Cry3 (Cry3Bb1, mCry3A, and eCry3.1Ab) hybrids led to concerns about durability and efficacy of products with single traits and of products containing a pyramid of a Cry3 protein and the binary Bt proteins Cry34Ab1 and Cry35Ab1. From 2012 to 2014, 43 field trials were conducted across the central United States to estimate root protection provided by plants expressing Cry34Ab1/Cry35Ab1 alone (Herculex RW) or pyramided with Cry3Bb1 (SmartStax). These technologies were evaluated with and without soil-applied insecticides to determine if additional management measures provided benefit where Cry3 performance was reduced. Trials were categorized for analysis based on rootworm damage levels on Cry3-expressing hybrids and rootworm feeding pressure within each trial. Across scenarios, Cry34Ab1/Cry35Ab1 hybrids provided excellent root protection. Pyramided traits provided greater root and yield protection than non-Bt plus a soil-applied insecticide, and only in trials where larval feeding pressure exceeded two nodes of damage did Cry34Ab1/Cry35Ab1 single-trait hybrids and pyramided hybrids show greater root protection from the addition of soil-applied insecticides. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

In situ surface transfer process of Cry1Ac protein on SiO2： The effect of biosurfactants for desorption.

PubMed

Miao, Shuzhou; Yuan, Xingzhong; Liang, Yunshan; Wang, Hou; Leng, Lijian; Wu, Zhibin; Jiang, Longbo; Li, Yifu; Mo, Dan; Zeng, Guangming

2018-01-05

Genetically modified Bacillus thuringiensis (Bt) crops, which have been widely used in agricultural transgenic plants, express insecticidal Cry proteins and release the toxin into soils. Taking into consideration the environmental risk of Cry proteins, biosurfactant-rhamnolipids were applied to desorb Cry proteins from soil environment, which has not been elucidated before. Quartz crystal microbalance with dissipation (QCM-D) was used in this article to investigate the adsorption and desorption behaviors of Cry1Ac on SiO 2 surface (model soil). Results showed that patch-controlled electrostatic attraction (PCEA) governed Cry1Ac adsorption to SiO 2, and the solution pH or ionic strength can affect PCEA. The adsorption kinetics could be fitted by the pseudo-second-order model, and the adsorption isotherm was fitted to Langmuir model with correlation coefficients higher than 0.999. The desorption characteristics of Cry1Ac from SiO 2 were assessed in the presence of mono-rhamnolipid, di-rhamnolipid or complex-rhamnolipid. Mono-rhamnolipid exhibited the most significant positive effect on desorption performance. With a complete removal of Cry1Ac reached when mono-rhamnolipid concentration was up to 50mgL -1 . Additionally, the desorption was enhanced at alkaline pH range, and Cry1Ac can be completely and rapidly desorbed by rhamnolipids from SiO 2 at ionic strength of 5×10 -2 M. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

PubMed Central

Pigott, Craig R.; Ellar, David J.

2007-01-01

Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

PubMed Central

Zhang, Fengjuan; Peng, Donghai; Cheng, Chunsheng; Zhou, Wei; Ju, Shouyong; Wan, Danfeng; Yu, Ziquan; Shi, Jianwei; Deng, Yaoyao; Wang, Fenshan; Ye, Xiaobo; Hu, Zhenfei; Lin, Jian; Ruan, Lifang; Sun, Ming

2016-01-01

Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control. PMID:26795495

Multiple assays indicate varying levels of cross resistance of Cry3Bb1-selected field populations of the western corn rootworm to mCry3A, eCry3.1Ab, and Cry34/35Ab1

USDA-ARS?s Scientific Manuscript database

Minnesota populations of the western corn rootworm (WCR) surviving Cry3Bb1-expressing corn in the field and WCR populations assumed to be susceptible to all Bt proteins were evaluated for susceptibility to Cry3Bb1, mCry3A, eCry3.1Ab, and Cry34/35Ab1 in diet assays and three different plant-based ass...

FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice.

PubMed

Hirano, Arisa; Braas, Daniel; Fu, Ying-Hui; Ptáček, Louis J

2017-04-11

The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Bacillus thuringiensis: A story of a successful bioinsecticide

PubMed Central

Bravo, Alejandra; Likitvivatanavong, Supaporn; Gill, Sarjeet S.; Soberón, Mario

2013-01-01

Bacillus thuringiensis (Bt) bacteria are insect pathogens that rely on insecticidal pore forming proteins known as Cry and Cyt toxins to kill their insect larval hosts. At least four different non-structurally related families of proteins form the Cry toxin group of toxins. The expression of certain Cry toxins in transgenic crops has contributed to an efficient control of insect pests resulting in a significant reduction in chemical insecticide use. The mode of action of the three domain Cry toxin family involves sequential interaction of these toxins with several insect midgut proteins facilitating the formation of a pre-pore oligomer structure and subsequent membrane insertion that leads to the killing of midgut insect cells by osmotic shock. In this manuscript we review recent progress in understanding the mode of action of this family of proteins in lepidopteran, dipteran and coleopteran insects. Interestingly, similar Cry-binding proteins have been identified in the three insect orders, as cadherin, aminopeptidase-N and alkaline phosphatase suggesting a conserved mode of action. Also, recent data on insect responses to Cry toxin attack is discussed. Finally, we review the different Bt based products, including transgenic crops, that are currently used in agriculture. PMID:21376122

Safety of Bacillus thuringiensis Cry1C protein for Daphnia magna based on different functional traits.

PubMed

Chen, Yi; Yang, Yan; Zhu, Haojun; Romeis, Jörg; Li, Yunhe; Peng, Yufa; Chen, Xiuping

2018-01-01

Cry1C is a Bacillus thuringiensis (Bt) insecticidal protein and it can be produced by transgenic rice lines developed in China. Cladocera species are common aquatic arthropods that may be exposed to insecticidal proteins produced in Bt-transgenic plants through ingestion of pollen or crop residues in water. As the cladoceran Daphnia magna plays an important role in the aquatic food chain, it is important to assess the possible effects of Bt crops to this species. To evaluate the safety of the Cry1C protein for D. magna, individuals were exposed to different concentrations of purified Cry1C protein in M4 medium for 21 days. Potassium dichromate (K 2 Cr 2 O 7 ), a known toxicant to D. magna, was added to M4 medium as a positive control treatment, and pure M4 medium was used as a negative control. Our results show that developmental, reproductive, and biochemical parameters of D. magna were not significantly different between Cry1C and negative control treatments but were significantly inhibited by the positive control. We thus conclude that D. magna is insensitive to Cry1C. Copyright © 2017 Elsevier Inc. All rights reserved.

40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

Code of Federal Regulations, 2014 CFR

2014-07-01

... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...

40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

Code of Federal Regulations, 2012 CFR

2012-07-01

... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...

40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

Code of Federal Regulations, 2011 CFR

2011-07-01

... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...

40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

Code of Federal Regulations, 2013 CFR

2013-07-01

... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...

40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

Code of Federal Regulations, 2010 CFR

2010-07-01

... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...

Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression

PubMed Central

Jakka, Siva R. K.; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J.; Narva, Kenneth; Blanco, Carlos A.

2015-01-01

Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae. PMID:26637593

Bt Crops Producing Cry1Ac, Cry2Ab and Cry1F Do Not Harm the Green Lacewing, Chrysoperla rufilabris

PubMed Central

Tian, Jun-Ce; Wang, Xiang-Ping; Long, Li-Ping; Romeis, Jörg; Naranjo, Steven E.; Hellmich, Richard L.; Wang, Ping; Earle, Elizabeth D.; Shelton, Anthony M.

2013-01-01

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms. PMID:23544126

Peptide selection and antibody generation for the prospective immunorecognition of Cry1Ab16 protein of transgenic maize.

PubMed

Costa, Joana; Marani, Mariela M; Grazina, Liliana; Villa, Caterina; Meira, Liliana; Oliveira, M Beatriz P P; Leite, José R S A; Mafra, Isabel

2017-09-15

The introduction of genes isolated from different Bacillus thuringiensis strains to express Cry-type toxins in transgenic crops is a common strategy to confer insect resistance traits. This work intended to extensively in silico analyse Cry1A(b)16 protein for the identification of peptide markers for the biorecognition of transgenic crops. By combining two different strategies based on several bioinformatic tools for linear epitope prediction, a set of seven peptides was successfully selected as potential Cry1A(b)16 immunogens. For the prediction of conformational epitopes, Cry1A(b)16 models were built on the basis of three independent templates of homologue proteins of Cry1A(a) and Cry1A(c) using an integrated approach. PcH_736-746 and PcH_876-886 peptides were selected as the best candidates, being synthesised and used for the production of polyclonal antibodies. To the best of our knowledge, this is the first attempt of selecting and defining linear peptides as immunogenic markers of Cry1A(b)-type toxins in transgenic maize. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evolution of Bacillus thuringiensis Cry toxins insecticidal activity.

PubMed

Bravo, Alejandra; Gómez, Isabel; Porta, Helena; García-Gómez, Blanca Ines; Rodriguez-Almazan, Claudia; Pardo, Liliana; Soberón, Mario

2013-01-01

Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize MON810 varieties for 90 days (EU 7th Framework Programme project GRACE).

PubMed

Tulinská, Jana; Adel-Patient, Karine; Bernard, Hervé; Líšková, Aurélia; Kuricová, Miroslava; Ilavská, Silvia; Horváthová, Mira; Kebis, Anton; Rollerová, Eva; Babincová, Júlia; Aláčová, Radka; Wal, Jean-Michel; Schmidt, Kerstin; Schmidtke, Jörg; Schmidt, Paul; Kohl, Christian; Wilhelm, Ralf; Schiemann, Joachim; Steinberg, Pablo

2018-07-01

The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insecticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various different animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadically altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance.

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis.

PubMed

Gao, Yulin; Hu, Yang; Fu, Qiang; Zhang, Jie; Oppert, Brenda; Lai, Fengxiang; Peng, Yufa; Zhang, Zhitao

2010-09-01

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC(50) of 0.24 and 0.30 microg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations. Copyright 2010 Elsevier Inc. All rights reserved.

Toxicity and mode of action of insecticidal Cry1A proteins from Bacillus thuringiensis in an insect cell line, CF-1.

PubMed

Portugal, Leivi; Gringorten, J Lawrence; Caputo, Guido F; Soberón, Mario; Muñoz-Garay, Carlos; Bravo, Alejandra

2014-03-01

Bacillus thuringiensis Cry toxins are insecticidal proteins used to control insect pests. The interaction of Cry toxins with the midgut of susceptible insects is a dynamic process involving activation of the toxin, binding to midgut receptors in the apical epithelium and conformational changes in the toxin molecule, leading to pore formation and cell lysis. An understanding of the molecular events underlying toxin mode of action is essential for the continued use of Cry toxins. In this work, we examined the mechanism of action of Cry1A toxins in the lepidopteran cell line CF-1, using native Cry1Ab and mutant forms of this protein that interfer with different steps in the mechanism of action, specifically, receptor binding, oligomerization or pore formation. These mutants lost activity against both Manduca sexta larvae and CF-1 cells. We also analyzed a mutation created in domain I of Cry1Ab, in which helix α-1 and part of helix α-2 were deleted (Cry1AbMod). Cry1AbMod is able to oligomerize in the absence of toxin receptors, and although it shows reduced activity against some susceptible insects, it kills insect pests that have developed resistance to native Cry1Ab. Cry1AbMod showed enhanced toxicity to CF-1, suggesting that oligomerization of native Cry1Ab may be a limiting step in its activity against CF-1 cells. The toxicity of Cry1Ac and Cry1AcMod were also analyzed. Our results suggest that some of the steps in the mode of action of Cry1A toxins are conserved in vivo in insect midgut cells and in vitro in an established cell line, CF-1. Copyright © 2013 Elsevier Inc. All rights reserved.

PRKCDBP (CAVIN3) and CRY2 associate with major depressive disorder.

PubMed

Kovanen, Leena; Donner, Kati; Kaunisto, Mari; Partonen, Timo

2017-01-01

Dysfunctions in the intrinsic clocks are suggested in patients with depressive disorders. The cryptochrome circadian clocks 1 and 2 (CRY1 and CRY2) proteins modulate circadian rhythms in a cell and influence emotional reactions and mood in an individual. The protein kinase C delta binding protein (PRKCDBP, or CAVIN3), similar to the serum deprivation response protein (SDPR, or CAVIN2), reduces metabolic stability of the PER2-CRY2 transcription factor complex that plays a role in the circadian rhythm synchronization. Our aim was to study SDPR, PRKCDBP, CRY1 and CRY2 genetic variants in depressive disorders. The sample included 5910 Finnish individuals assessed with the Munich-Composite International Diagnostic Interview (M-CIDI) in year 2000. In year 2011, 3424 individuals were assessed again. After genotype quality control, there were 383 subjects with major depressive disorder, 166 with dysthymia, and 479 with depressive disorders (major depressive disorder, dysthymia or both), and 4154 healthy controls. A total of 48 single-nucleotide polymorphisms from SDPR, PRKCDBP, CRY1 and CRY2 genes were analyzed using logistic regression models controlling for age and gender. The earlier reported association of CRY2 variants with dysthymia was confirmed and extended to major depressive disorder (q<0.05). In addition, novel associations of PRKCDBP rs1488864 with depressive disorders (q=0.02) and with major depressive disorder in specific (q=0.007) were found. The number of cases was moderate and coverage of PRKCDB was limited. CRY2 and PRKCDBP variants may be risk factors of major depressive disorder and provide information for diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Consumption of Bt Rice Pollen Containing Cry1C or Cry2A Protein Poses a Low to Negligible Risk to the Silkworm Bombyx mori (Lepidoptera: Bombyxidae)

PubMed Central

Yang, Yan; Liu, Yue; Cao, Fengqin; Chen, Xiuping; Cheng, Lisheng; Romeis, Jörg; Li, Yunhe; Peng, Yufa

2014-01-01

By consuming mulberry leaves covered with pollen from nearby genetically engineered, insect-resistant rice lines producing Cry proteins derived from Bacillus thuringiensis (Bt), larvae of the domestic silkworm, Bombyx mori (Linnaeus) (Lepidoptera: Bombyxidae), could be exposed to insecticidal proteins. Laboratory experiments were conducted to assess the potential effects of Cry1C- or Cry2A-producing transgenic rice (T1C-19, T2A-1) pollen on B. mori fitness. In a short-term assay, B. mori larvae were fed mulberry leaves covered with different densities of pollen from Bt rice lines or their corresponding near isoline (control) for the first 3 d and then were fed mulberry leaves without pollen. No effect was detected on any life table parameter, even at 1800 pollen grains/cm2 leaf, which is much higher than the mean natural density of rice pollen on leaves of mulberry trees near paddy fields. In a long-term assay, the larvae were fed Bt and control pollen in the same way but for their entire larval stage (approximately 27 d). Bt pollen densities ≥150 grains/cm2 leaf reduced 14-d larval weight, increased larval development time, and reduced adult eclosion rate. ELISA analyses showed that 72.6% of the Cry protein was still detected in the pollen grains excreted with the feces. The low exposure of silkworm larvae to Cry proteins when feeding Bt rice pollen may be the explanation for the relatively low toxicity detected in the current study. Although the results demonstrate that B. mori larvae are sensitive to Cry1C and Cry2A proteins, the exposure levels that harmed the larvae in the current study are far greater than natural exposure levels. We therefore conclude that consumption of Bt rice pollen will pose a low to negligible risk to B. mori. PMID:25014054

Does Cry1Ab protein affect learning performances of the honey bee Apis mellifera L. (Hymenoptera, Apidae)?

PubMed

Ramirez-Romero, R; Desneux, N; Decourtye, A; Chaffiol, A; Pham-Delègue, M H

2008-06-01

Genetically modified Bt crops are increasingly used worldwide but side effects and especially sublethal effects on beneficial insects remain poorly studied. Honey bees are beneficial insects for natural and cultivated ecosystems through pollination. The goal of the present study was to assess potential effects of two concentrations of Cry1Ab protein (3 and 5000 ppb) on young adult honey bees. Following a complementary bioassay, our experiments evaluated effects of the Cry1Ab on three major life traits of young adult honey bees: (a) survival of honey bees during sub-chronic exposure to Cry1Ab, (b) feeding behaviour, and (c) learning performance at the time that honey bees become foragers. The latter effect was tested using the proboscis extension reflex (PER) procedure. The same effects were also tested using a chemical pesticide, imidacloprid, as positive reference. The tested concentrations of Cry1Ab protein did not cause lethal effects on honey bees. However, honey bee feeding behaviour was affected when exposed to the highest concentration of Cry1Ab protein, with honey bees taking longer to imbibe the contaminated syrup. Moreover, honey bees exposed to 5000 ppb of Cry1Ab had disturbed learning performances. Honey bees continued to respond to a conditioned odour even in the absence of a food reward. Our results show that transgenic crops expressing Cry1Ab protein at 5000 ppb may affect food consumption or learning processes and thereby may impact honey bee foraging efficiency. The implications of these results are discussed in terms of risks of transgenic Bt crops for honey bees.

LRIG2 Mutations Cause Urofacial Syndrome

PubMed Central

Stuart, Helen M.; Roberts, Neil A.; Burgu, Berk; Daly, Sarah B.; Urquhart, Jill E.; Bhaskar, Sanjeev; Dickerson, Jonathan E.; Mermerkaya, Murat; Silay, Mesrur Selcuk; Lewis, Malcolm A.; Olondriz, M. Beatriz Orive; Gener, Blanca; Beetz, Christian; Varga, Rita E.; Gülpınar, Ömer; Süer, Evren; Soygür, Tarkan; Özçakar, Zeynep B.; Yalçınkaya, Fatoş; Kavaz, Aslı; Bulum, Burcu; Gücük, Adnan; Yue, Wyatt W.; Erdogan, Firat; Berry, Andrew; Hanley, Neil A.; McKenzie, Edward A.; Hilton, Emma N.; Woolf, Adrian S.; Newman, William G.

2013-01-01

Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease. We have previously shown that UFS is also caused by mutations in HPSE2, encoding heparanase-2. LRIG2 and heparanase-2 were immunodetected in nerve fascicles growing between muscle bundles within the human fetal bladder, directly implicating both molecules in neural development in the lower urinary tract. PMID:23313374

Performance and cross-crop resistance of Cry1F-maize selected Spodoptera frugiperda on transgenic Bt cotton: implications for resistance management

PubMed Central

Yang, Fei; Kerns, David L.; Brown, Sebe; Kurtz, Ryan; Dennehy, Tim; Braxton, Bo; Head, Graham; Huang, Fangneng

2016-01-01

Transgenic crops producing Bacillus thuringiensis (Bt) proteins have become a primary tool in pest management. Due to the intensive use of Bt crops, resistance of the fall armyworm, Spodoptera frugiperda, to Cry1F maize has occurred in Puerto Rico, Brazil, and some areas of the southeastern U.S. The sustainability of Bt crops faces a great challenge because the Cry1F-maize resistant S. frugiperda may also infest other Bt crops in multiple cropping ecosystems. Here we examined the survival and plant injury of a S. frugiperda population selected with Cry1F maize on three single-gene and five pyramided Bt cotton products. Larvae of Cry1F-susceptible (SS), -heterozygous (RS), and -resistant (RR) genotypes of S. frugiperda were all susceptible to the pyramided cotton containing Cry1Ac/Cry2Ab, Cry1Ac/Cry1F/Vip3A, Cry1Ab/Cry2Ae, or Cry1Ab/Cry2Ae/Vip3A, and the single-gene Cry2Ae cotton. Pyramided cotton containing Cry1Ac/Cry1F was effective against SS and RS, but not for RR. These findings show that the Cry1F-maize selected S. frugiperda can cause cross-crop resistance to other Bt crops expressing similar insecticidal proteins. Resistance management and pest management programs that utilize diversify mortality factors must be implemented to ensure the sustainability of Bt crops. This is especially important in areas where resistance to single-gene Bt crops is already widespread. PMID:27301612

Performance and cross-crop resistance of Cry1F-maize selected Spodoptera frugiperda on transgenic Bt cotton: implications for resistance management.

PubMed

Yang, Fei; Kerns, David L; Brown, Sebe; Kurtz, Ryan; Dennehy, Tim; Braxton, Bo; Head, Graham; Huang, Fangneng

2016-06-15

Transgenic crops producing Bacillus thuringiensis (Bt) proteins have become a primary tool in pest management. Due to the intensive use of Bt crops, resistance of the fall armyworm, Spodoptera frugiperda, to Cry1F maize has occurred in Puerto Rico, Brazil, and some areas of the southeastern U.S. The sustainability of Bt crops faces a great challenge because the Cry1F-maize resistant S. frugiperda may also infest other Bt crops in multiple cropping ecosystems. Here we examined the survival and plant injury of a S. frugiperda population selected with Cry1F maize on three single-gene and five pyramided Bt cotton products. Larvae of Cry1F-susceptible (SS), -heterozygous (RS), and -resistant (RR) genotypes of S. frugiperda were all susceptible to the pyramided cotton containing Cry1Ac/Cry2Ab, Cry1Ac/Cry1F/Vip3A, Cry1Ab/Cry2Ae, or Cry1Ab/Cry2Ae/Vip3A, and the single-gene Cry2Ae cotton. Pyramided cotton containing Cry1Ac/Cry1F was effective against SS and RS, but not for RR. These findings show that the Cry1F-maize selected S. frugiperda can cause cross-crop resistance to other Bt crops expressing similar insecticidal proteins. Resistance management and pest management programs that utilize diversify mortality factors must be implemented to ensure the sustainability of Bt crops. This is especially important in areas where resistance to single-gene Bt crops is already widespread.

First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize.

PubMed

Fantozzi, Anna; Ermolli, Monica; Marini, Massimiliano; Scotti, Domenico; Balla, Branko; Querci, Maddalena; Langrell, Stephen R H; Van den Eede, Guy

2007-02-21

An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

PubMed

Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

2013-03-01

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. Copyright © 2012 Elsevier Inc. All rights reserved.

Mutational disruption of the ABCC2 gene in fall armyworm, Spodoptera frugiperda, confers resistance to the Cry1Fa and Cry1A.105 insecticidal proteins.

PubMed

Flagel, Lex; Lee, Young Wha; Wanjugi, Humphrey; Swarup, Shilpa; Brown, Alana; Wang, Jinling; Kraft, Edward; Greenplate, John; Simmons, Jeni; Adams, Nancy; Wang, Yanfei; Martinelli, Samuel; Haas, Jeffrey A; Gowda, Anilkumar; Head, Graham

2018-05-08

The use of Bt proteins in crops has revolutionized insect pest management by offering effective season-long control. However, field-evolved resistance to Bt proteins threatens their utility and durability. A recent example is field-evolved resistance to Cry1Fa and Cry1A.105 in fall armyworm (Spodoptera frugiperda). This resistance has been detected in Puerto Rico, mainland USA, and Brazil. A S. frugiperda population with suspected resistance to Cry1Fa was sampled from a maize field in Puerto Rico and used to develop a resistant lab colony. The colony demonstrated resistance to Cry1Fa and partial cross-resistance to Cry1A.105 in diet bioassays. Using genetic crosses and proteomics, we show that this resistance is due to loss-of-function mutations in the ABCC2 gene. We characterize two novel mutant alleles from Puerto Rico. We also find that these alleles are absent in a broad screen of partially resistant Brazilian populations. These findings confirm that ABCC2 is a receptor for Cry1Fa and Cry1A.105 in S. frugiperda, and lay the groundwork for genetically enabled resistance management in this species, with the caution that there may be several distinct ABCC2 resistances alleles in nature.

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action.

PubMed

Jurat-Fuentes, Juan Luis; Crickmore, Neil

2017-01-01

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are used as active components of biopesticides and as plant incorporated protectants in transgenic crops. One of the most relevant attributes of these Bt protein-based insecticidal technologies is their high specificity, which assures lack of detrimental effects on non-target insects, vertebrates and the environment. The identification of specificity determinants in Bt insecticidal proteins could guide risk assessment for novel insecticidal proteins currently considered for commercialization. In this work we review the available data on specificity determinants of crystal (Cry) insecticidal proteins as the Bt toxins most well characterized and used in transgenic crops. The multi-step mode of action of the Cry insecticidal proteins allows various factors to potentially affect specificity determination and here we define seven levels that could influence specificity. The relative relevance of each of these determinants on efficacy of transgenic crops producing Cry insecticidal proteins is also discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Multi-state trials of Bt sweet corn varieties for control of the corn earworm (Lepidoptera: Noctuidae).

PubMed

Shelton, A M; Olmstead, D L; Burkness, E C; Hutchison, W D; Dively, G; Welty, C; Sparks, A N

2013-10-01

Field tests in 2010-2011 were performed in New York, Minnesota, Maryland, Ohio, and Georgia to compare Bt sweet corn lines expressing Cry1A.105 + Cry2Ab2 and Cry1Ab with their non-Bt isolines, with and without the use of foliar insecticides. The primary insect pest in all locations during the trial years was Heliocoverpa zea (Boddie), which is becoming the most serious insect pest of sweet corn in the United States. At harvest, the ears were measured for marketability according to fresh market and processing standards. For fresh market and processing, least squares regression showed significant effects of protein expression, state, and insecticide frequency. There was a significant effect of year for fresh market but not for processing. The model also showed significant effects of H. zea per ear by protein expression. Sweet corn containing two genes (Cry1A.105 + Cry2Ab2) and a single gene (Cry1Ab) provided high marketability, and both Bt varieties significantly outperformed the traditional non-Bt isolines in nearly all cases regardless of insecticide application frequency. For pest suppression of H. zea, plants expressing Bt proteins consistently performed better than non-Bt isoline plants, even those sprayed at conventional insecticide frequencies. Where comparisons in the same state were made between Cry1A.105 + Cry2Ab2 and Cry1Ab plants for fresh market, the product expressing Cry1A.105 + Cry2Ab2 provided better protection and resulted in less variability in control. Overall, these results indicate Cry1A.105 + Cry2Ab2 and Cry1Ab plants are suitable for fresh market and processing corn production across a diversity of growing regions and years. Our results demonstrate that Bt sweet corn has the potential to significantly reduce the use of conventional insecticides against lepidopteran pests and, in turn, reduce occupational and environmental risks that arise from intensive insecticide use.

Agronomic performance of insect-protected and herbicide-tolerant MON 89034 × TC1507 × NK603 × DAS-40278-9 corn is equivalent to that of conventional corn.

PubMed

de Cerqueira, Denise T Rezende; Schafer, Ariane C; Fast, Brandon J; Herman, Rod A

2017-07-03

Agronomic characteristics of genetically modified (GM) MON 89034 × TC1507 × NK603 × DAS-40278-9 (PowerCore™ Enlist™), MON 89034 × TC1507 × NK603 (PowerCore™), and DAS-40278-9 (Enlist™) corn, a non-GM near-isogenic hybrid, and 2 commercial non-GM hybrids were assessed in a field study to determine if the agronomic performance of the GM corn hybrids is equivalent to that of non-transgenic hybrid corn. The MON 89034 × TC1507 × NK603 × DAS-40278-9 hybrid corn was developed through stacking of 4 individual transgenic events, MON 89034, TC1507, NK603, and DAS-40278-9 by traditional breeding and contains the cry1A.105 and cry2Ab2 (MON 89034), cry1F and pat (TC1507), cp4 epsps (NK603) and aad-1 (DAS-40278-9) transgenes. These transgenes encode the proteins Cry1A.105, Cry2Ab2, and Cry1F, which confer insect resistance, PAT, CP4 EPSPS, and AAD-1, which confer herbicide tolerance. The following agronomic characteristics were assessed in the study: initial and final stand count, seedling vigor, time to silk, time to pollen shed, pollen viability, plant height, ear height, stalk lodging, root lodging, days to maturity, stay green, disease incidence, insect damage, herbicide injury, and yield. The agronomic assessment was conducted in 2 regions of Brazil (Indianopolis-MG; Cravinhos-SP). The agronomic attributes for all GM entries were statistically indistinguishable from the non-GM near-isogenic hybrid. In addition, most of the agronomic assessments fell within the range of the commercial varieties included in the study. Taken together, MON 89034 × TC1507 × NK603 × DAS-40278, MON 89034 × TC1507 × NK603, and DAS-40278-9 were found to be agronomically equivalent to non-GM corn.

Investigation of the Cry4B-prohibitin interaction in Aedes aegypti cells.

PubMed

Kuadkitkan, Atichat; Smith, Duncan R; Berry, Colin

2012-10-01

Bacillus thuringiensis (Bt) produces insecticidal toxins active against insects. Cry4B, one of the major insecticidal toxins produced by Bt subsp. israelensis, is highly toxic to mosquitoes in the genus Aedes: the major vectors of dengue, yellow fever, and chikungunya. Previous work has shown that Cry4B binds to several mid-gut membrane proteins in Aedes aegypti larvae including prohibitin, a protein recently identified as a receptor that also mediates entry of dengue virus into Aedes cells. This study confirms the interaction between Cry4B and prohibitin by co-immunoprecipitation analysis and demonstrates colocalization of prohibitin and Cry4B by confocal microscopy. While activated Cry4B toxin showed high larvicidal activity, it was not cytotoxic to two Aedes cell lines, allowing determination of its effect on dengue virus infectivity in the absence of Cry4B-induced cell lysis. Pre-exposure of Aedes cells to Cry4B resulted in a significant reduction in the number of infected cells compared to untreated cells.

Occurrence of maize detritus and a transgenic insecticidal protein (Cry1Ab) within the stream network of an agricultural landscape

PubMed Central

Tank, Jennifer L.; Rosi-Marshall, Emma J.; Royer, Todd V.; Whiles, Matt R.; Griffiths, Natalie A.; Frauendorf, Therese C.; Treering, David J.

2010-01-01

Widespread planting of maize throughout the agricultural Midwest may result in detritus entering adjacent stream ecosystems, and 63% of the 2009 US maize crop was genetically modified to express insecticidal Cry proteins derived from Bacillus thuringiensis. Six months after harvest, we conducted a synoptic survey of 217 stream sites in Indiana to determine the extent of maize detritus and presence of Cry1Ab protein in the stream network. We found that 86% of stream sites contained maize leaves, cobs, husks, and/or stalks in the active stream channel. We also detected Cry1Ab protein in stream-channel maize at 13% of sites and in the water column at 23% of sites. We found that 82% of stream sites were adjacent to maize fields, and Geographical Information Systems analyses indicated that 100% of sites containing Cry1Ab-positive detritus in the active stream channel had maize planted within 500 m during the previous crop year. Maize detritus likely enters streams throughout the Corn Belt; using US Department of Agriculture land cover data, we estimate that 91% of the 256,446 km of streams/rivers in Iowa, Illinois, and Indiana are located within 500 m of a maize field. Maize detritus is common in low-gradient stream channels in northwestern Indiana, and Cry1Ab proteins persist in maize leaves and can be measured in the water column even 6 mo after harvest. Hence, maize detritus, and associated Cry1Ab proteins, are widely distributed and persistent in the headwater streams of a Corn Belt landscape. PMID:20876106

Occurrence of maize detritus and a transgenic insecticidal protein (Cry1Ab) within the stream network of an agricultural landscape.

PubMed

Tank, Jennifer L; Rosi-Marshall, Emma J; Royer, Todd V; Whiles, Matt R; Griffiths, Natalie A; Frauendorf, Therese C; Treering, David J

2010-10-12

Widespread planting of maize throughout the agricultural Midwest may result in detritus entering adjacent stream ecosystems, and 63% of the 2009 US maize crop was genetically modified to express insecticidal Cry proteins derived from Bacillus thuringiensis. Six months after harvest, we conducted a synoptic survey of 217 stream sites in Indiana to determine the extent of maize detritus and presence of Cry1Ab protein in the stream network. We found that 86% of stream sites contained maize leaves, cobs, husks, and/or stalks in the active stream channel. We also detected Cry1Ab protein in stream-channel maize at 13% of sites and in the water column at 23% of sites. We found that 82% of stream sites were adjacent to maize fields, and Geographical Information Systems analyses indicated that 100% of sites containing Cry1Ab-positive detritus in the active stream channel had maize planted within 500 m during the previous crop year. Maize detritus likely enters streams throughout the Corn Belt; using US Department of Agriculture land cover data, we estimate that 91% of the 256,446 km of streams/rivers in Iowa, Illinois, and Indiana are located within 500 m of a maize field. Maize detritus is common in low-gradient stream channels in northwestern Indiana, and Cry1Ab proteins persist in maize leaves and can be measured in the water column even 6 mo after harvest. Hence, maize detritus, and associated Cry1Ab proteins, are widely distributed and persistent in the headwater streams of a Corn Belt landscape.

Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control

PubMed Central

Bravo, Alejandra; Gill, Sarjeet S.; Soberón, Mario

2007-01-01

Bacillus thuringiensis Cry and Cyt protein families are a diverse group of proteins with activity against insects of different orders - Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores. Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane. Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect pests and insect vectors of human diseases. PMID:17198720

Baseline sensitivity of lepidopteran corn pests in India to Cry1Ab insecticidal protein of Bacillus thuringiensis.

PubMed

Jalali, Sushil K; Lalitha, Yadavalli; Kamath, Subray P; Mohan, Komarlingam S; Head, Graham P

2010-08-01

Genetically engineered corn (Bt corn) expressing Bacillus thuringiensis Berliner insecticidal protein Cry1Ab is a biotechnological option being considered for management of lepidopteran corn pests in India. As a resistance management practice it was essential to determine the sensitivity of multiple populations of the stalk borer Chilo partellus (Swinhoe), pink borer Sesamia inferens (Walker) and the cob borer Helicoverpa armigera (Hübner) to Cry1Ab protein through bioassays. The insect populations were collected during growing seasons of Rabi 2005 (October 2005 to February 2006) and Kharif 2006 (May to September 2006). Multiple populations of the three lepidopteran corn pests were found to be susceptible to Cry1Ab. Median lethal concentrations (LC(50)) ranged between 0.008 and 0.068 microg Cry1Ab mL(-1) diet for 18 populations of C. partellus (across two seasons), between 0.12 and 1.99 microg mL(-1) for seven populations of H. armigera and between 0.46 and 0.56 microg mL(-1) for two populations of S. inferens. Dose-response concentrations for lethality and growth inhibition have been determined to mark baseline sensitivity of multiple populations of key lepidopteran corn pests in India to Cry1Ab protein. These benchmark values will be referenced while monitoring resistance to Cry1Ab should Bt corn hybrids expressing Cry1Ab be approved for commercial cultivation in India. Copyright (c) 2010 Society of Chemical Industry.

High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigers to Bt Soybean in Brazil.

PubMed

Dourado, Patrick M; Bacalhau, Fabiana B; Amado, Douglas; Carvalho, Renato A; Martinelli, Samuel; Head, Graham P; Omoto, Celso

2016-01-01

The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F2 screen. The mean Cry1Ac lethal concentration (LC50) ranged from 0.11 to 1.82 μg·mL-1 of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F2 family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil.

DDB1-Mediated CRY1 Degradation Promotes FOXO1-Driven Gluconeogenesis in Liver.

PubMed

Tong, Xin; Zhang, Deqiang; Charney, Nicholas; Jin, Ethan; VanDommelen, Kyle; Stamper, Kenneth; Gupta, Neil; Saldate, Johnny; Yin, Lei

2017-10-01

Targeted protein degradation through ubiquitination is an important step in the regulation of glucose metabolism. Here, we present evidence that the DDB1-CUL4A ubiquitin E3 ligase functions as a novel metabolic regulator that promotes FOXO1-driven hepatic gluconeogenesis. In vivo, hepatocyte-specific Ddb1 deletion leads to impaired hepatic gluconeogenesis in the mouse liver but protects mice from high-fat diet-induced hyperglycemia. Lack of Ddb1 downregulates FOXO1 protein expression and impairs FOXO1-driven gluconeogenic response. Mechanistically, we discovered that DDB1 enhances FOXO1 protein stability via degrading the circadian protein cryptochrome 1 (CRY1), a known target of DDB1 E3 ligase. In the Cry1 depletion condition, insulin fails to reduce the nuclear FOXO1 abundance and suppress gluconeogenic gene expression. Chronic depletion of Cry1 in the mouse liver not only increases FOXO1 protein but also enhances hepatic gluconeogenesis. Thus, we have identified the DDB1-mediated CRY1 degradation as an important target of insulin action on glucose homeostasis. © 2017 by the American Diabetes Association.

Efficacy of genetically modified Bt toxins alone and in combinations against pink bollworm resistant to Cry1Ac and Cry2Ab

USDA-ARS?s Scientific Manuscript database

Evolution of resistance in pests threatens the long-term success of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and ...

Application of Box-Behnken experimental design to optimize the extraction of insecticidal Cry1Ac from soil.

PubMed

Li, Yan-Liang; Fang, Zhi-Xiang; You, Jing

2013-02-20

A validated method for analyzing Cry proteins is a premise to study the fate and ecological effects of contaminants associated with genetically engineered Bacillus thuringiensis crops. The current study has optimized the extraction method to analyze Cry1Ac protein in soil using a response surface methodology with a three-level-three-factor Box-Behnken experimental design (BBD). The optimum extraction conditions were at 21 °C and 630 rpm for 2 h. Regression analysis showed a good fit of the experimental data to the second-order polynomial model with a coefficient of determination of 0.96. The method was sensitive and precise with a method detection limit of 0.8 ng/g dry weight and relative standard deviations at 7.3%. Finally, the established method was applied for analyzing Cry1Ac protein residues in field-collected soil samples. Trace amounts of Cry1Ac protein were detected in the soils where transgenic crops have been planted for 8 and 12 years.

A Novel Tenebrio molitor Cadherin Is a Functional Receptor for Bacillus thuringiensis Cry3Aa Toxin*

PubMed Central

Fabrick, Jeff; Oppert, Cris; Lorenzen, Marcé D.; Morris, Kaley; Oppert, Brenda; Jurat-Fuentes, Juan Luis

2009-01-01

Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepidopteran cadherin B. thuringiensis receptors. A peptide containing the putative toxin binding region from TmCad1 bound specifically to Cry3Aa and promoted the formation of Cry3Aa toxin oligomers, proposed to be mediators of toxicity in lepidopterans. Injection of TmCad1-specific double-stranded RNA into T. molitor larvae resulted in knockdown of the TmCad1 transcript and conferred resistance to Cry3Aa toxicity. These data demonstrate the functional role of TmCad1 as a Cry3Aa receptor in T. molitor and reveal similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera. PMID:19416969

Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests.

PubMed

Chen, Wen-Bo; Lu, Guo-Qing; Cheng, Hong-Mei; Liu, Chen-Xi; Xiao, Yu-Tao; Xu, Chao; Shen, Zhi-Cheng; Wu, Kong-Ming

2017-10-01

Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for integrated pest management in China. Copyright © 2017 Elsevier Inc. All rights reserved.

Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize.

PubMed

Roda, A; Mirasoli, M; Guardigli, M; Michelini, E; Simoni, P; Magliulo, M

2006-03-01

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL(-1) Cry1Ab (3 or 5 pg mL(-1), depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation.

Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds.

PubMed

Wang, Qing; Zhu, Yi; Sun, Lin; Li, Lebin; Jin, Shuangxia; Zhang, Xianlong

2016-02-01

A promoter of the PNZIP (Pharbitis nil leucine zipper) gene (1.459 kb) was cloned from Pharbitis nil and fused to the GUS (β-glucuronidase) and Bacillus thuringiensis endotoxin (Cry9C) genes. Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP::GUS cotton was very faint. Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus (CaMV) 35S::Cry9C line were selected for enzyme-linked immunosorbent assay (ELISA) and insect bioassays. Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 μg g(-1) fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP::Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 μg g(-1) fresh weight respectively. In contrast, seeds of the PNZIP::Cry9C line (PZ1.3) accumulated only 0.26 μg g(-1) fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S::Cry9C line. The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology.

Unlinked genetic loci control the reduced transcription of aminopeptidase N 1 and 3 in the European corn borer and determine tolerance to Bacillus thuringiensis Cry1Ab toxin

USDA-ARS?s Scientific Manuscript database

Crystalline (Cry) toxins from Bacillus thuringiensis (Bt) control insect feeding damage on crop plants via foliar applications or by expression within transgenic plants, but continued Bt use is threatened by the buildup of insect resistance traits. Aminopeptidase N (apn) gene family members encode m...

Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

PubMed Central

Chakroun, Maissa; Banyuls, Núria; Bel, Yolanda; Escriche, Baltasar

2016-01-01

SUMMARY Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet. PMID:26935135

Comprehensive in silico allergenicity assessment of novel protein engineered chimeric Cry proteins for safe deployment in crops.

PubMed

Rathinam, Maniraj; Singh, Shweta; Pattanayak, Debasis; Sreevathsa, Rohini

2017-08-02

Development of chimeric Cry toxins by protein engineering of known and validated proteins is imperative for enhancing the efficacy and broadening the insecticidal spectrum of these genes. Expression of novel Cry proteins in food crops has however created apprehensions with respect to the safety aspects. To clarify this, premarket evaluation consisting of an array of analyses to evaluate the unintended effects is a prerequisite to provide safety assurance to the consumers. Additionally, series of bioinformatic tools as in silico aids are being used to evaluate the likely allergenic reaction of the proteins based on sequence and epitope similarity with known allergens. In the present study, chimeric Cry toxins developed through protein engineering were evaluated for allergenic potential using various in silico algorithms. Major emphasis was on the validation of allergenic potential on three aspects of paramount significance viz., sequence-based homology between allergenic proteins, validation of conformational epitopes towards identification of food allergens and physico-chemical properties of amino acids. Additionally, in vitro analysis pertaining to heat stability of two of the eight chimeric proteins and pepsin digestibility further demonstrated the non-allergenic potential of these chimeric toxins. The study revealed for the first time an all-encompassing evaluation that the recombinant Cry proteins did not show any potential similarity with any known allergens with respect to the parameters generally considered for a protein to be designated as an allergen. These novel chimeric proteins hence can be considered safe to be introgressed into plants.

Analysis of Homologs of Cry-toxin Receptor-Related Proteins in the Midgut of a Non-Bt Target, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

PubMed Central

Shao, Ensi; Lin, Li; Liu, Sijun; Zhang, Jiao; Chen, Xuelin; Sha, Li; Huang, Zhipeng; Huang, Biwang; Guan, Xiong

2018-01-01

Abstract The brown planthopper (BPH) Nilaparvata lugens is one of the most destructive insect pests in the rice fields of Asia. Like other hemipteran insects, BPH is not susceptible to Cry toxins of Bacillus thuringiensis (Bt) or transgenic rice carrying Bt cry genes. Lack of Cry receptors in the midgut is one of the main reasons that BPH is not susceptible to the Cry toxins. The main Cry-binding proteins (CBPs) of the susceptible insects are cadherin, aminopeptidase N (APN), and alkaline phosphatase (ALP). In this study, we analyzed and validated de novo assembled transcripts from transcriptome sequencing data of BPH to identify and characterize homologs of cadherin, APN, and ALP. We then compared the cadherin-, APN-, and ALP-like proteins of BPH to previously reported CBPs to identify their homologs in BPH. The sequence analysis revealed that at least one cadherin, one APN, and two ALPs of BPH contained homologous functional domains identified from the Cry-binding cadherin, APN, and ALP, respectively. Quantitative real-time polymerase chain reaction used to verify the expression level of each putative Cry receptor homolog in the BPH midgut indicated that the CBPs homologous APN and ALP were expressed at high or medium-high levels while the cadherin was expressed at a low level. These results suggest that homologs of CBPs exist in the midgut of BPH. However, differences in key motifs of CBPs, which are functional in interacting with Cry toxins, may be responsible for insusceptibility of BPH to Cry toxins. PMID:29415259

Cytotoxicity and binding profiles of activated Cry1Ac and Cry2Ab to three insect cell lines

USDA-ARS?s Scientific Manuscript database

While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (ATP-binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baselin...

Is the Cry1Ab protein from Bacillus thuringiensis (Bt) taken up by plants from soils previously planted with Bt corn and by carrot from hydroponic culture?

PubMed

Icoz, I; Andow, D; Zwahlen, C; Stotzky, G

2009-07-01

The uptake of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) by various crops from soils on which Bt corn had previously grown was determined. In 2005, the Cry1Ab protein was detected by Western blot in tissues (leaves plus stems) of basil, carrot, kale, lettuce, okra, parsnip, radish, snap bean, and soybean but not in tissues of beet and spinach and was estimated by enzyme-linked immunosorbent assay (ELISA) to be 0.05 +/- 0.003 ng g(-1) of fresh plant tissue in basil, 0.02 +/- 0.014 ng g(-1) in okra, and 0.34 +/- 0.176 ng g(-1) in snap bean. However, the protein was not detected by ELISA in carrot, kale, lettuce, parsnip, radish, and soybean or in the soils by Western blot. In 2006, the Cry1Ab protein was detected by Western blot in tissues of basil, carrot, kale, radish, snap bean, and soybean from soils on which Bt corn had been grown the previous year and was estimated by ELISA to be 0.02 +/- 0.014 ng g(-1) of fresh plant tissue in basil, 0.19 +/- 0.060 ng g(-1) in carrot, 0.05 +/- 0.018 ng g(-1) in kale, 0.04 +/- 0.022 ng g(-1) in radish, 0.53 +/- 0.170 ng g(-1) in snap bean, and 0.15 +/- 0.071 ng g(-1) in soybean. The Cry1Ab protein was also detected by Western blot in tissues of basil, carrot, kale, radish, and snap bean but not of soybean grown in soil on which Bt corn had not been grown since 2002; the concentration was estimated by ELISA to be 0.03 +/- 0.021 ng g(-1) in basil, 0.02 +/- 0.008 ng g(-1) in carrot, 0.04 +/- 0.017 ng g(-1) in kale, 0.02 +/- 0.012 ng g(-1) in radish, 0.05 +/- 0.004 ng g(-1) in snap bean, and 0.09 +/- 0.015 ng g(-1) in soybean. The protein was detected by Western blot in 2006 in most soils on which Bt corn had or had not been grown since 2002. The Cry1Ab protein was detected by Western blot in leaves plus stems and in roots of carrot after 56 days of growth in sterile hydroponic culture to which purified Cry1Ab protein had been added and was estimated by ELISA to be 0.08 +/- 0.021 and 0.60 +/- 0.148 ng g(-1) of fresh leaves plus stems and roots, respectively. No Cry1Ab protein was detected in the tissues of carrot grown in hydroponic culture to which no Cry1Ab protein had been added. Because of the different results obtained with different commercial Western blot (i.e., from Envirologix and Agdia) and ELISA kits (i.e., from Envirologix, Agdia, and Abraxis), it is not clear whether the presence of the Cry1Ab protein in the tissues of some plants under field condition and in carrot in sterile hydroponic culture was the result of the uptake of the protein by the plants or of the accuracy and sensitivity of the different commercial kits used. More detailed studies with additional techniques are obviously needed to confirm the uptake of Cry proteins from soil by plants subsequently planted after a Bt crop.

Establishment of a dietary exposure assay for evaluating the toxicity of insecticidal compounds to Apolygus lucorum (Hemiptera: Miridae).

PubMed

Zhao, Man; Li, Yunhe; Yuan, Xiangdong; Liang, Gemei; Wang, Bingjie; Liu, Chen; Khaing, Myint Myint

2018-06-01

With the commercialization of transgenic cotton that expresses Bt (Bacillus thuringiensis) insecticidal proteins, mirid bugs have become key pests in cotton and maize fields in China. Genetically engineered (GE) crops for controlling mirids are unavailable owing to a lack of suitable insecticidal genes. In this study, we developed and validated a dietary exposure assay for screening insecticidal compounds and for assessing the potential effects of insecticidal proteins produced by GE plants on Apolygus lucorum, one of the main mirid pests of Bt cotton and Bt maize. Diets containing potassium arsenate (PA) or the cysteine protease inhibitor E-64 were used as positive controls for validating the efficacy of the dietary exposure assay. The results showed that with increasing concentrations of PA or E-64, A. lucorum larval development time was prolonged and adult weight and fecundity were decreased, suggesting that the dietary exposure assay was useful for detecting the toxicity of insecticidal compounds to A. lucorum. This assay was then used to assess the toxicity of Cry1Ab, Cry1Ac, Cry1F, Cry2Aa, and Cry2Ab proteins, which have been transformed into several crops, against A. lucorum. The results showed that A. lucorum did not show a negative effect by feeding on an artificial diet containing any of the purified Cry proteins. No significant changes in the activities of digestive, detoxifying, or antioxidant enzymes were detected in A. lucorum that fed on a diet containing Cry proteins, but A. lucorum fitness was reduced when the insect fed on a diet containing E-64 or PA. These results demonstrate that A. lucorum is not sensitive to the tested Cry proteins and that the dietary exposure assay is useful for evaluating the toxicity of insecticidal compounds to this species. Copyright © 2018 Elsevier Ltd. All rights reserved.

Field-Evolved Resistance in Corn Earworm to Cry Proteins Expressed by Transgenic Sweet Corn.

PubMed

Dively, Galen P; Venugopal, P Dilip; Finkenbinder, Chad

2016-01-01

Transgenic corn engineered with genes expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Berliner) (Bt) are now a major tool in insect pest management. With its widespread use, insect resistance is a major threat to the sustainability of the Bt transgenic technology. For all Bt corn expressing Cry toxins, the high dose requirement for resistance management is not achieved for corn earworm, Helicoverpa zea (Boddie), which is more tolerant to the Bt toxins. We present field monitoring data using Cry1Ab (1996-2016) and Cry1A.105+Cry2Ab2 (2010-2016) expressing sweet corn hybrids as in-field screens to measure changes in field efficacy and Cry toxin susceptibility to H. zea. Larvae successfully damaged an increasing proportion of ears, consumed more kernel area, and reached later developmental stages (4th - 6th instars) in both types of Bt hybrids (Cry1Ab-event Bt11, and Cry1A.105+Cry2Ab2-event MON89034) since their commercial introduction. Yearly patterns of H. zea population abundance were unrelated to reductions in control efficacy. There was no evidence of field efficacy or tissue toxicity differences among different Cry1Ab hybrids that could contribute to the decline in control efficacy. Supportive data from laboratory bioassays demonstrate significant differences in weight gain and fitness characteristics between the Maryland H. zea strain and a susceptible strain. In bioassays with Cry1Ab expressing green leaf tissue, Maryland H. zea strain gained more weight than the susceptible strain at all concentrations tested. Fitness of the Maryland H. zea strain was significantly lower than that of the susceptible strain as indicated by lower hatch rate, longer time to adult eclosion, lower pupal weight, and reduced survival to adulthood. After ruling out possible contributing factors, the rapid change in field efficacy in recent years and decreased susceptibility of H. zea to Bt sweet corn provide strong evidence of field-evolved resistance in H. zea populations to multiple Cry toxins. The high adoption rate of Bt field corn and cotton, along with the moderate dose expression of Cry1Ab and related Cry toxins in these crops, and decreasing refuge compliance probably contributed to the evolution of resistance. Our results have important implications for resistance monitoring, refuge requirements and other regulatory policies, cross-resistance issues, and the sustainability of the pyramided Bt technology.

The tiered-evaluation of the effects of transgenic cry1c rice on Cyrtorhinus lividipennis, a main predator of Nilaparvata lugens

PubMed Central

Han, Yu; Ma, Fugang; Nawaz, Muhammad; Wang, Yu; Cai, Wanlun; Zhao, Jing; He, Yueping; Hua, Hongxia; Zou, Yulan

2017-01-01

T1C-19, a newly developed transgenic cry1C rice line, expresses cry1C under the control of the maize ubiquitin promoter, and is highly resistant to lepidopteran pests of rice. Cyrtorhinus lividipennis is the major predator of the eggs and young nymphs of Nilaparvata lugens, which is the main non-target sap-sucking insect pest of Bt rice. C. lividipennis may be exposed to Cry1C protein, thus biosafety evaluations of transgenic cry1C rice on C. lividipennis should be conducted before the commercialization of T1C-19. In the current study, we tested the direct toxicity of elevated doses of Cry1C to C. lividipennis, effects of T1C-19 on the life-table parameters of C. lividipennis via preying planthoppers, and effects of T1C-19 on the population density and dynamics in rice fields. No detrimental effects on development, survival, female ratio and body weight of C. lividipennis were caused by direct exposure to elevated doses of the Cry1C protein or prey-mediated exposure to realistic doses of the protein. The population density and dynamics did not significantly differ between C. lividipennis in T1C-19 and non-transgenic rice fields. Thus, transgenic cry1C rice had no negative effects on C. lividipennis. This is the first report of the effects of transgenic cry1C rice on C. lividipennis. PMID:28205641

The tiered-evaluation of the effects of transgenic cry1c rice on Cyrtorhinus lividipennis, a main predator of Nilaparvata lugens

NASA Astrophysics Data System (ADS)

Han, Yu; Ma, Fugang; Nawaz, Muhammad; Wang, Yu; Cai, Wanlun; Zhao, Jing; He, Yueping; Hua, Hongxia; Zou, Yulan

2017-02-01

T1C-19, a newly developed transgenic cry1C rice line, expresses cry1C under the control of the maize ubiquitin promoter, and is highly resistant to lepidopteran pests of rice. Cyrtorhinus lividipennis is the major predator of the eggs and young nymphs of Nilaparvata lugens, which is the main non-target sap-sucking insect pest of Bt rice. C. lividipennis may be exposed to Cry1C protein, thus biosafety evaluations of transgenic cry1C rice on C. lividipennis should be conducted before the commercialization of T1C-19. In the current study, we tested the direct toxicity of elevated doses of Cry1C to C. lividipennis, effects of T1C-19 on the life-table parameters of C. lividipennis via preying planthoppers, and effects of T1C-19 on the population density and dynamics in rice fields. No detrimental effects on development, survival, female ratio and body weight of C. lividipennis were caused by direct exposure to elevated doses of the Cry1C protein or prey-mediated exposure to realistic doses of the protein. The population density and dynamics did not significantly differ between C. lividipennis in T1C-19 and non-transgenic rice fields. Thus, transgenic cry1C rice had no negative effects on C. lividipennis. This is the first report of the effects of transgenic cry1C rice on C. lividipennis.

Parallel processing on the Livermore VAX 11/780-4 parallel processor system with compatibility to Cray Research, Inc. (CRI) multitasking. Version 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werner, N.E.; Van Matre, S.W.

1985-05-01

This manual describes the CRI Subroutine Library and Utility Package. The CRI library provides Cray multitasking functionality on the four-processor shared memory VAX 11/780-4. Additional functionality has been added for more flexibility. A discussion of the library, utilities, error messages, and example programs is provided.

Comparative Proteomic and Nutritional Composition Analysis of Independent Transgenic Pigeon Pea Seeds Harboring cry1AcF and cry2Aa Genes and Their Nontransgenic Counterparts.

PubMed

Mishra, Pragya; Singh, Shweta; Rathinam, Maniraj; Nandiganti, Muralimohan; Ram Kumar, Nikhil; Thangaraj, Arulprakash; Thimmegowda, Vinutha; Krishnan, Veda; Mishra, Vagish; Jain, Neha; Rai, Vandna; Pattanayak, Debasis; Sreevathsa, Rohini

2017-02-22

Safety assessment of genetically modified plants is an important aspect prior to deregulation. Demonstration of substantial equivalence of the transgenics compared to their nontransgenic counterparts can be performed using different techniques at various molecular levels. The present study is a first-ever comprehensive evaluation of pigeon pea transgenics harboring two independent cry genes, cry2Aa and cry1AcF. The absence of unintended effects in the transgenic seed components was demonstrated by proteome and nutritional composition profiling. Analysis revealed that no significant differences were found in the various nutritional compositional analyses performed. Additionally, 2-DGE-based proteome analysis of the transgenic and nontransgenic seed protein revealed that there were no major changes in the protein profile, although a minor fold change in the expression of a few proteins was observed. Furthermore, the study also demonstrated that neither the integration of T-DNA nor the expression of the cry genes resulted in the production of unintended effects in the form of new toxins or allergens.

Edible Safety Assessment of Genetically Modified Rice T1C-1 for Sprague Dawley Rats through Horizontal Gene Transfer, Allergenicity and Intestinal Microbiota.

PubMed

Zhao, Kai; Ren, Fangfang; Han, Fangting; Liu, Qiwen; Wu, Guogan; Xu, Yan; Zhang, Jian; Wu, Xiao; Wang, Jinbin; Li, Peng; Shi, Wei; Zhu, Hong; Lv, Jianjun; Zhao, Xiao; Tang, Xueming

2016-01-01

In this study, assessment of the safety of transgenic rice T1C-1 expressing Cry1C was carried out by: (1) studying horizontal gene transfer (HGT) in Sprague Dawley rats fed transgenic rice for 90 d; (2) examining the effect of Cry1C protein in vitro on digestibility and allergenicity; and (3) studying the changes of intestinal microbiota in rats fed with transgenic rice T1C-1 in acute and subchronic toxicity tests. Sprague Dawley rats were fed a diet containing either 60% GM Bacillus thuringiensis (Bt) rice T1C-1 expressing Cry1C protein, the parental rice Minghui 63, or a basic diet for 90 d. The GM Bt rice T1C-1 showed no evidence of HGT between rats and transgenic rice. Sequence searching of the Cry1C protein showed no homology with known allergens or toxins. Cry1C protein was rapidly degraded in vitro with simulated gastric and intestinal fluids. The expressed Cry1C protein did not induce high levels of specific IgG and IgE antibodies in rats. The intestinal microbiota of rats fed T1C-1 was also analyzed in acute and subchronic toxicity tests by DGGE. Cluster analysis of DGGE profiles revealed significant individual differences in the rats' intestinal microbiota.

Bacillus thuringiensis plants expressing Cry1Ac, Cry2Ab and Cry1F are not toxic to the assassin bug, Zelus renardii

USDA-ARS?s Scientific Manuscript database

Cotton and maize delivering insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non-target organisms, especially natural enemies that suppress pest populations. In th...

Arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication

USDA-ARS?s Scientific Manuscript database

Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well ch...

No impact of transgenic cry1C rice on the rove beetle Paederus fuscipes, a generalist predator of brown planthopper Nilaparvata lugens

NASA Astrophysics Data System (ADS)

Meng, Jiarong; Mabubu, Juma Ibrahim; Han, Yu; He, Yueping; Zhao, Jing; Hua, Hongxia; Feng, Yanni; Wu, Gang

2016-07-01

T1C-19 is newly developed transgenic rice active against lepidopteran pests, and expresses a synthesized cry1C gene driven by the maize ubiquitin promoter. The brown planthopper, Nilaparvata lugens, is a major non-target pest of rice, and the rove beetle (Paederus fuscipes) is a generalist predator of N. lugens nymphs. As P. fuscipes may be exposed to the Cry1C protein through preying on N. lugens, it is essential to assess the potential effects of transgenic cry1C rice on this predator. In this study, two experiments (a direct feeding experiment and a tritrophic experiment) were conducted to evaluate the ecological risk of cry1C rice to P. fuscipes. No significant negative effects were observed in the development, survival, female ratio and body weight of P. fuscipes in both treatments of direct exposure to elevated doses of Cry1C protein and prey-mediated exposure to realistic doses of the protein. This indicated that cry1C rice had no detrimental effects on P. fuscipes. This work represents the first study of an assessment continuum for the effects of transgenic cry1C rice on P. fuscipes. Use of the rove beetle as an indicator species to assess potential effects of genetically modified crops on non-target arthropods is feasible.

No impact of transgenic cry1C rice on the rove beetle Paederus fuscipes, a generalist predator of brown planthopper Nilaparvata lugens

PubMed Central

Meng, Jiarong; Mabubu, Juma Ibrahim; Han, Yu; He, Yueping; Zhao, Jing; Hua, Hongxia; Feng, Yanni; Wu, Gang

2016-01-01

T1C-19 is newly developed transgenic rice active against lepidopteran pests, and expresses a synthesized cry1C gene driven by the maize ubiquitin promoter. The brown planthopper, Nilaparvata lugens, is a major non-target pest of rice, and the rove beetle (Paederus fuscipes) is a generalist predator of N. lugens nymphs. As P. fuscipes may be exposed to the Cry1C protein through preying on N. lugens, it is essential to assess the potential effects of transgenic cry1C rice on this predator. In this study, two experiments (a direct feeding experiment and a tritrophic experiment) were conducted to evaluate the ecological risk of cry1C rice to P. fuscipes. No significant negative effects were observed in the development, survival, female ratio and body weight of P. fuscipes in both treatments of direct exposure to elevated doses of Cry1C protein and prey-mediated exposure to realistic doses of the protein. This indicated that cry1C rice had no detrimental effects on P. fuscipes. This work represents the first study of an assessment continuum for the effects of transgenic cry1C rice on P. fuscipes. Use of the rove beetle as an indicator species to assess potential effects of genetically modified crops on non-target arthropods is feasible. PMID:27444416

Crystallin-αB Regulates Skeletal Muscle Homeostasis via Modulation of Argonaute2 Activity*

PubMed Central

Neppl, Ronald L.; Kataoka, Masaharu; Wang, Da-Zhi

2014-01-01

The core functional machinery of the RNAi pathway is the RNA-induced silencing complex (RISC), wherein Argonaute2 (Ago2) is essential for siRNA-directed endonuclease activity and RNAi/microRNA-mediated gene silencing. Crystallin-αB (CryAB) is a small heat shock protein involved in preventing protein aggregation. We demonstrate that CryAB interacts with the N and C termini of Ago2, not the catalytic site defined by the convergence of the PAZ, MID, and PIWI domains. We further demonstrate significantly reduced Ago2 activity in the absence of CryAB, highlighting a novel role of CryAB in the mammalian RNAi/microRNA pathway. In skeletal muscle of CryAB null mice, we observe a shift in the hypertrophy-atrophy signaling axis toward atrophy under basal conditions. Moreover, loss of CryAB altered the capability of satellite cells to regenerate skeletal muscle. These studies establish that CryAB is necessary for normal Ago2/RISC activity and cellular homeostasis in skeletal muscle. PMID:24782307

KPNB1 mediates PER/CRY nuclear translocation and circadian clock function.

PubMed

Lee, Yool; Jang, A Reum; Francey, Lauren J; Sehgal, Amita; Hogenesch, John B

2015-08-29

Regulated nuclear translocation of the PER/CRY repressor complex is critical for negative feedback regulation of the circadian clock of mammals. However, the precise molecular mechanism is not fully understood. Here, we report that KPNB1, an importin β component of the ncRNA repressor of nuclear factor of activated T cells (NRON) ribonucleoprotein complex, mediates nuclear translocation and repressor function of the PER/CRY complex. RNAi depletion of KPNB1 traps the PER/CRY complex in the cytoplasm by blocking nuclear entry of PER proteins in human cells. KPNB1 interacts mainly with PER proteins and directs PER/CRY nuclear transport in a circadian fashion. Interestingly, KPNB1 regulates the PER/CRY nuclear entry and repressor function, independently of importin α, its classical partner. Moreover, inducible inhibition of the conserved Drosophila importin β in lateral neurons abolishes behavioral rhythms in flies. Collectively, these data show that KPNB1 is required for timely nuclear import of PER/CRY in the negative feedback regulation of the circadian clock.

Influence of calcareous soil on Cry3Bb1 expression and efficacy in the field.

PubMed

Wangila, David S; Valencia J, Arnubio; Wang, Haichuan; Siegfried, Blair D; Meinke, Lance J

2017-06-01

Greater than expected injury by western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) to Cry3Bb1 expressing maize hybrids (Zea mays L.) has been reported in southwestern Nebraska. Affected areas of some fields are often associated with high pH calcareous soils where maize growth is poor and iron chlorosis is common. As part of a comprehensive study to understand potential causes of unexpected injury, experiments were conducted during 2013 and 2014 to ascertain whether the calcareous soil conditions and associated poor maize growth negatively affect the expression of Cry3Bb1. Quantitative determination of Cry3Bb1 protein expression levels in root tissues was carried out on plants at V5-V6 growth stage using the enzyme-linked immunosorbent assay. Cry3Bb1 and non-Bt near isoline maize hybrids were artificially infested with Cry3Bb1-susceptible WCR eggs to measure survival and efficacy of Cry3Bb1 maize in calcareous and non-calcareous soils. Results showed that there was not a significant difference in expression of Cry3Bb1 protein between plants from calcareous and non-calcareous soils (18.9-21.2 µg/g fresh weight). Western corn rootworm survival was about sevenfold greater from the non-Bt isoline than Cry3Bb1 maize indicating that Cry3Bb1 performed as expected when infested with a Cry3Bb1 susceptible rootworm population. When survival from calcareous and non-calcareous soils was compared, no significant differences were observed in each soil. A significant positive correlation between soil pH and expression of Cry3Bb1 protein in roots was detected from samples collected in 2014 but not in 2013. No such correlation was found between soil pH and survival of WCR. Results suggest that Cry3Bb1 expression levels were sufficient to provide adequate root protection against WCR regardless of soil environment, indicating that lowered Cry3Bb1 expression is not a contributing factor to the greater than expected WCR injury observed in some southwestern Nebraska maize fields.

77 FR 26547 - Amendment/Extension of an Experimental Use Permit

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-04

...-incorporated protectant (PIP) [Event 5307] Bacillus thuringiensis eCry3.1Ab protein and the genetic material necessary for its production (vector pSYN12274) in Event 5307 corn (SYN-[Oslash]53[Oslash]7-1) and combined and single trait hybrids with one or more of the following additional PIPs: (1) [Bt11] Bacillus...

Drosophila small heat shock protein CryAB ensures structural integrity of developing muscles, and proper muscle and heart performance.

PubMed

Wójtowicz, Inga; Jabłońska, Jadwiga; Zmojdzian, Monika; Taghli-Lamallem, Ouarda; Renaud, Yoan; Junion, Guillaume; Daczewska, Malgorzata; Huelsmann, Sven; Jagla, Krzysztof; Jagla, Teresa

2015-03-01

Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. Here, we show that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). Our data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryAB(R120G), which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, we demonstrate that CryAB ensures myofibrillar integrity in Drosophila muscles during development and propose that it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture. © 2015. Published by The Company of Biologists Ltd.

Aquatic degradation of Cry1Ab protein and decomposition dynamics of transgenic corn leaves under controlled conditions.

PubMed

Böttger, Rita; Schaller, Jörg; Lintow, Sven; Gert Dudel, E

2015-03-01

The increasing cultivation of genetically modified corn plants (Zea mays) during the last decades is suggested as a potential risk to the environment. One of these genetically modified variety expressed the insecticidal Cry1Ab protein originating from Bacillus thuringiensis (Bt), resulting in resistance against Ostrinia nubilalis, the European corn borer. Transgenic litter material is extensively studied regarding the decomposition in soils. However, only a few field studies analyzed the fate of the Cry1Ab protein and the impact of green and senescent leaf litter from corn on the decomposition rate and related ecosystem functions in aquatic environments. Consequently, a microbial litter decomposition experiment was conducted under controlled semi-natural conditions in batch culture using two maize varieties: one variety with Cry1Ab and another one with the appertaining Iso-line as control treatment. The results showed no significant differences between the treatment with Cry1Ab and the Iso-line regarding loss of total mass in dry weight of 43% for Iso-line and 45% for Bt-corn litter, lignin content increased to 137.5% (Iso-line) and 115.7% (Bt-corn), and phenol loss decreased by 53.6% (Iso-line), 62.2% (Bt-corn) during three weeks of the experiment. At the end of the experiment Cry1Ab protein was still detected with 6% of the initial concentration. A slightly but significant lower cellulose content was found for the Cry1Ab treatment compared to the Iso-line litter at the end of the experiment. The significant higher total protein (25%) and nitrogen (25%) content in Bt corn, most likely due to the additionally expression of the transgenic protein, may increase the microbial cellulose degradation and decrease microbial lignin degradation. In conclusion a relevant year by year input of protein and therefore nitrogen rich Bt corn litter into aquatic environments may affect the balanced nutrient turnover in aquatic ecosystems. Copyright © 2014 Elsevier Inc. All rights reserved.

75 FR 34040 - Bacillus thuringiensis

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-16

... protein in corn under the FFDCA. The temporary tolerance exemption expires on June 1, 2012. DATES: This..., and III (Refs. 3, 5, 6 and 7). The toxin portions of Cry proteins are characterized by having five... rates. 3. Prevalence in food. Preliminary expression level analysis shows that the eCry.1Ab protein is...

Transgenic rice expressing the cry2AX1 gene confers resistance to multiple lepidopteran pests.

PubMed

Chakraborty, M; Reddy, P Sairam; Mustafa, G; Rajesh, G; Narasu, V M Laxmi; Udayasuriyan, V; Rana, Debashis

2016-10-01

A chimeric Bacillus thuringiensis toxin (Bt) gene, cry2AX1was cloned in a bi-selectable marker free binary vector construct. The cry2AX1 gene, driven by the Chrysanthemum rbcS1 promoter, was introduced into JK1044R, the restorer line (Oryza sativa L. ssp. Indica) of a notified commercially grown rice hybrid in India, by Agrobacterium-mediated transformation. Its effect against two major lepidopteran insect pests viz., yellow stem borer (YSB) Scirpophaga incertulas, rice leaf folder (RLF) Cnaphalocrocis medinalis and one minor insect pest, oriental army worm (OAW) Mythimna separata was demonstrated through bioassays of transgenic rice plants under laboratory and greenhouse conditions. The rbcS1 promoter with chloroplast signal peptide was used to avoid Cry2AX1 protein expression in rice seed endosperm tissue. A total of 37 independent transformants were generated, of which after preliminary molecular characterization and YSB bioassay screening, five events were selected for their protein expression and bioefficacy against all three rice insect. One elite transgenic rice line, BtE15, was identified with Cry2AX1 expression ranging from 0.68 to 1.34 µg g(-1) leaf fresh weight and with 80-92 % levels of resistance against rice pests at the vegetative and reproductive stages. Increase in Cry2AX1 protein concentration was also observed with crop maturity. The Cry2AX1protein concentration in the de-husked seeds was negligible (as low as 2.7-3.6 ng g(-1)). These results indicate the potential application of cry2AX1 gene in rice for protection against YSB, RLF and OAW.

Vaccinia-related kinase 3 (VRK3) sets the circadian period and amplitude by affecting the subcellular localization of clock proteins in mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Nayoung; Department of Brain Science, Ajou University School of Medicine, 164 Worldcup-ro, Yeongtong-gu, Suwon, Kyunggi-do, 16499; Song, Jieun

In the eukaryotic circadian clock machinery, negative feedback repression of CLOCK (CLK) and BMAL1 transcriptional activity by PERIOD (PER) and CRYPTOCHROME (CRY) underlies the basis for 24 h rhythmic gene expression. Thus, precise regulation of the time-dependent nuclear entry of circadian repressors is crucial to generating normal circadian rhythms. Here, we sought to identify novel kinase(s) that regulate nuclear entry of mammalian CRY1 (mCRY1) with an unbiased screening using red fluorescent protein (RFP)-tagged human kinome expression plasmids in mammalian cells. Transient expression of human vaccinia-related kinase 3 (hVRK3) reduced the nuclear presence of mCRY1. hVRK3 expression also induced alterations in themore » subcellular localization of other core clock proteins, including mCRY2, mPER2, and BMAL1. In contrast, the subcellular localization of mCLK was not changed. Given that singly expressed mCLK mostly resides in the cytoplasm and that nuclear localization sequence (NLS) mutation of hVRK3 attenuated the effect of hVRK3 co-expression on subcellular localization, ectopically expressed hVRK3 presumably reduces the retention of proteins in the nucleus. Finally, downregulation of hvrk3 using siRNA reduced the amplitude and lengthened the period of the cellular bioluminescence rhythm. Taken together, these data suggest that VRK3 plays a role in setting the amplitude and period length of circadian rhythms in mammalian cells. - Highlights: • Screening was performed to identify kinases that regulate CRY1 subcellular localization. • VRK3 alters the subcellular localization of CRY1, CRY2, PER2, and BMAL1. • VRK3 knock-down alters the circadian bioluminescence rhythm in mammalian cells.« less

Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformans.

PubMed

Eigenheer, Richard A; Jin Lee, Young; Blumwald, Eduardo; Phinney, Brett S; Gelli, Angie

2007-06-01

Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or beta-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography-mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans.

Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects

PubMed Central

Kota, Madhuri; Daniell, Henry; Varma, Sam; Garczynski, Stephen F.; Gould, Fred; Moar, William J.

1999-01-01

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000–10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field. PMID:10051556

Cryptochrome 2 expression level is critical for adrenocorticotropin stimulation of cortisol production in the capuchin monkey adrenal.

PubMed

Torres-Farfan, C; Abarzua-Catalan, L; Valenzuela, F J; Mendez, N; Richter, H G; Valenzuela, G J; Serón-Ferré, M

2009-06-01

Timely production of glucocorticoid hormones in response to ACTH is essential for survival by coordinating energy intake and expenditure and acting as homeostatic regulators against stress. Adrenal cortisol response to ACTH is clock time dependent, suggesting that an intrinsic circadian oscillator in the adrenal cortex contributes to modulate the response to ACTH. Circadian clock gene expression has been reported in the adrenal cortex of several species. However, there are no reports accounting for potential involvement of adrenal clock proteins on cortisol response to ACTH. Here we explored whether the clock protein cryptochrome 2 (CRY2) knockdown modifies the adrenal response to ACTH in a primate. Adrenal gland explants from adult capuchin monkey (n = 5) were preincubated for 6 h with transfection vehicle (control) or with two different Cry2 antisense and sense probes followed by 48 h incubation in medium alone (no ACTH) or with 100 nm ACTH. Under control and sense conditions, ACTH increased cortisol production, whereas CRY2 suppression inhibited ACTH-stimulated cortisol production. Expression of the steroidogenic enzymes steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase at 48 h of incubation was increased by ACTH in control explants and suppressed by Cry2 knockdown. Additionally, we found that Cry2 knockdown decreased the expression of the clock gene brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) at the mRNA and protein levels. Altogether these results strongly support that the clock protein CRY2 is involved in the mechanism by which ACTH increases the expression of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase. Thus, adequate expression levels of components of the adrenal circadian clock are required for an appropriate cortisol response to ACTH.

The diverse nematicidal properties and biocontrol efficacy of Bacillus thuringiensis Cry6A against the root-knot nematode Meloidogyne hapla.

PubMed

Yu, Ziquan; Xiong, Jing; Zhou, Qiaoni; Luo, Haiyan; Hu, Shengbiao; Xia, Liqiu; Sun, Ming; Li, Lin; Yu, Ziniu

2015-02-01

Cry6A toxin from Bacillus thuringiensis is a representative nematicidal crystal protein with a variety of nematicidal properties to free-living nematode Caenorhabditis elegans. Cry6A shares very low homology and different structure with Cry5B, another representative nematicidal crystal protein, and probably acts in a distinct pathway. All these strongly indicate that Cry6A toxin is likely a potent candidate for nematicide. The present study dealt with global investigation to determine the detrimental impacts of Cry6Aa2 toxin on Meloidogyne hapla, a root-knot nematode, and evaluated its biocontrol efficacy in pot experiment. Obtained results indicated that Cry6Aa2 toxin exhibits obvious toxicity to second-stage juvenile of M. hapla, and significantly inhibits egg hatch, motility, and penetration to host plant. Pot experiment suggested that soil drenching with spore-crystal mixture of Cry6Aa2 can clearly lighten the disease of root-knot nematode, including reduction of galling index and egg masses on host plant root, decreasing final population of nematode in soil. Moreover, application of Cry6Aa2 can obviously promote plant growth. These results demonstrated that Cry6Aa2 toxin is a promising nematicidal agent, and possesses great potential in plant-parasitic nematode management and construction of transgenic crop with constant resistance to nematode. Copyright © 2015 Elsevier Inc. All rights reserved.

Bt crops producing Cry1Ac, Cry2Ab and Cry1F do not harm the green lacewing, chrysoperla rufilabris

USDA-ARS?s Scientific Manuscript database

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Le...

Cry8Ca2-containing layer-by-layer microcapsules for the pH-controlled release of crystal protein.

PubMed

Li, Feng; Yan, Yue; Wang, Dandan; Zhang, Jie; Guo, Shuyuan

2014-01-01

To extend the activity of crystal proteins by protection from environmental stress, we developed a new type of microcapsule containing Cry8Ca2 protoxins. Layer-by-layer (LbL) microcapsules containing Cry8Ca2 were successfully prepared for the first time by the alternate deposition of poly(acrylic acid) (PAH) and Cry8Ca2 at pH 6 on the surface of poly(styrene sulphonate) (PSS)-doped CaCO3 microbeads. Scanning electron microscopy (SEM) photos showed that microparticles were spherical in shape, approximately 2 μm in diameter. After removing the templates, the loading results were observed with a confocal laser scattering microscope (CLSM) by using fluorescein-labelled Cry8Ca2. The Cry8Ca2 protoxins were released from the microcapsules when they were exposed to a pH higher than 6 due to the loss of the electrostatic attraction. The microcapsules displayed resistance to proteinase K. Bioassay result demonstrated that the microcapsules with Cry8Ca2 displayed approximately equivalent insecticidal activity to the larvae of Anomala corpulenta compared to the free Cry8Ca2.

Hypothesis driven single cell dual oscillator mathematical model of circadian rhythms

PubMed Central

S, Shiju

2017-01-01

Molecular mechanisms responsible for 24 h circadian oscillations, entrainment to external cues, encoding of day length and the time-of-day effects have been well studied experimentally. However, it is still debated from the molecular network point of view whether each cell in suprachiasmatic nuclei harbors two molecular oscillators, where one tracks dawn and the other tracks dusk activities. A single cell dual morning and evening oscillator was proposed by Daan et al., based on the molecular network that has two sets of similar non-redundant per1/cry1 and per2/cry2 circadian genes and each can independently maintain their endogenous oscillations. Understanding of dual oscillator dynamics in a single cell at molecular level may provide insight about the circadian mechanisms that encodes day length variations and its response to external zeitgebers. We present here a realistic dual oscillator model of circadian rhythms based on the series of hypotheses proposed by Daan et al., in which they conjectured that the circadian genes per1/cry1 track dawn while per2/cry2 tracks dusk and they together constitute the morning and evening oscillators (dual oscillator). Their hypothesis also provides explanations about the encoding of day length in terms of molecular mechanisms of per/cry expression. We frame a minimal mathematical model with the assumption that per1 acts a morning oscillator and per2 acts as an evening oscillator and to support and interpret this assumption we fit the model to the experimental data of per1/per2 circadian temporal dynamics, phase response curves (PRC's), and entrainment phenomena under various light-dark conditions. We also capture different patterns of splitting phenomena by coupling two single cell dual oscillators with neuropeptides vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP) as the coupling agents and provide interpretation for the occurrence of splitting in terms of ME oscillators, though they are not required to explain the morning and evening oscillators. The proposed dual oscillator model based on Daan's hypothesis supports per1 and per2 playing the role of morning and evening oscillators respectively and this may be the first step towards the understanding of the core molecular mechanism responsible for encoding the day length. PMID:28486525

Hypothesis driven single cell dual oscillator mathematical model of circadian rhythms.

PubMed

S, Shiju; Sriram, K

2017-01-01

Molecular mechanisms responsible for 24 h circadian oscillations, entrainment to external cues, encoding of day length and the time-of-day effects have been well studied experimentally. However, it is still debated from the molecular network point of view whether each cell in suprachiasmatic nuclei harbors two molecular oscillators, where one tracks dawn and the other tracks dusk activities. A single cell dual morning and evening oscillator was proposed by Daan et al., based on the molecular network that has two sets of similar non-redundant per1/cry1 and per2/cry2 circadian genes and each can independently maintain their endogenous oscillations. Understanding of dual oscillator dynamics in a single cell at molecular level may provide insight about the circadian mechanisms that encodes day length variations and its response to external zeitgebers. We present here a realistic dual oscillator model of circadian rhythms based on the series of hypotheses proposed by Daan et al., in which they conjectured that the circadian genes per1/cry1 track dawn while per2/cry2 tracks dusk and they together constitute the morning and evening oscillators (dual oscillator). Their hypothesis also provides explanations about the encoding of day length in terms of molecular mechanisms of per/cry expression. We frame a minimal mathematical model with the assumption that per1 acts a morning oscillator and per2 acts as an evening oscillator and to support and interpret this assumption we fit the model to the experimental data of per1/per2 circadian temporal dynamics, phase response curves (PRC's), and entrainment phenomena under various light-dark conditions. We also capture different patterns of splitting phenomena by coupling two single cell dual oscillators with neuropeptides vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP) as the coupling agents and provide interpretation for the occurrence of splitting in terms of ME oscillators, though they are not required to explain the morning and evening oscillators. The proposed dual oscillator model based on Daan's hypothesis supports per1 and per2 playing the role of morning and evening oscillators respectively and this may be the first step towards the understanding of the core molecular mechanism responsible for encoding the day length.

Analysis of flour and food samples for cry9C from bioengineered corn.

PubMed

Orlandi, Palmer A; Lampel, Keith A; South, Paul K; Assar, Samir K; Carter, Laurenda; Levy, Dan D

2002-02-01

StarLink corn is a variety of yellow corn that has been genetically modified by the insertion of an altered cry9C gene into the plant genome. resulting in expression of the insecticidal Cry9C protein. The U.S. Environmental Protection Agency has approved StarLink corn for use in animal feed but not in food intended for human consumption. Therefore, under the U.S. Food, Drug, and Cosmetic Act, any food intended for human consumption in which the presence of StarLink corn is indicated by the presence of either the Cry9C protein or the cry9C gene would be considered adulterated. Extraction and PCR-based methods were used to detect the presence of the cry9C DNA initially in corn flour and corn meal, and then these methods were extended to the analysis of processed corn products, including taco shells, cereals, baby foods, party snacks, and chips, for the presence of this modified genetic material. In a survey of 63 products, the cry9C transgene was detected in 4 taco shells.

Bt crops benefit natural enemies to control non-target pests

PubMed Central

Tian, Jun-Ce; Yao, Ju; Long, Li-Ping; Romeis, Jörg; Shelton, Anthony M.

2015-01-01

Crops producing insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) control important lepidopteran pests. However, pests such as aphids not susceptible to Cry proteins may require other integrated pest management (IPM) tactics, including biological control. We fed aphids on Bt and non-Bt plants and analyzed the Bt protein residue in aphids and compared the effects of Bt plants and a pyrethroid, lambda-cyhalothrin, on the performance of three natural enemies (predators: Coleomegilla maculata and Eupeodes americanus; parasitoid Aphidius colemani) of the green peach aphid, Myzus persicae. No Bt protein residues in aphids were detected and no significant differences were recorded in the performance of pyrethroid-resistant aphids that fed on Bt broccoli expressing Cry1Ab or Cry1C, or on non-Bt broccoli plants treated or not treated with the pyrethroid. This indicated the aphids were not affected by the Cry proteins or the pyrethroid, thus removing any effect of prey quality. Tri-trophic experiments demonstrated that no C. maculata and E. americanus survived consumption of pyrethroid-treated aphids and that ovipositional behavior of A. colemani was impaired when provided with pyrethroid-treated aphids. In contrast, natural enemies were not affected when fed aphids reared on Bt broccoli, thus demonstrating the safety of these Bt plants for IPM. PMID:26559133

Bt crops benefit natural enemies to control non-target pests.

PubMed

Tian, Jun-Ce; Yao, Ju; Long, Li-Ping; Romeis, Jörg; Shelton, Anthony M

2015-11-12

Crops producing insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) control important lepidopteran pests. However, pests such as aphids not susceptible to Cry proteins may require other integrated pest management (IPM) tactics, including biological control. We fed aphids on Bt and non-Bt plants and analyzed the Bt protein residue in aphids and compared the effects of Bt plants and a pyrethroid, lambda-cyhalothrin, on the performance of three natural enemies (predators: Coleomegilla maculata and Eupeodes americanus; parasitoid Aphidius colemani) of the green peach aphid, Myzus persicae. No Bt protein residues in aphids were detected and no significant differences were recorded in the performance of pyrethroid-resistant aphids that fed on Bt broccoli expressing Cry1Ab or Cry1C, or on non-Bt broccoli plants treated or not treated with the pyrethroid. This indicated the aphids were not affected by the Cry proteins or the pyrethroid, thus removing any effect of prey quality. Tri-trophic experiments demonstrated that no C. maculata and E. americanus survived consumption of pyrethroid-treated aphids and that ovipositional behavior of A. colemani was impaired when provided with pyrethroid-treated aphids. In contrast, natural enemies were not affected when fed aphids reared on Bt broccoli, thus demonstrating the safety of these Bt plants for IPM.

Protein quality control in protection against systolic overload cardiomyopathy: the long term role of small heat shock proteins

PubMed Central

Kumarapeli, Asangi R K; Horak, Kathleen; Wang, Xuejun

2010-01-01

Molecular chaperones represent the first line of defense of intracellular protein quality control. As a major constituent of molecular chaperones, heat shock proteins (HSP) are known to confer cardiomyocyte short-term protection against various insults and injuries. Previously, we reported that the small HSP αB-crystallin (CryAB) attenuates cardiac hypertrophic response in mice subjected to 2 weeks of severe pressure overload. However, the long-term role of small HSPs in cardiac hypertrophy and failure has rarely been studied. The present study investigates the cardiac responses to chronic severe pressure overload in CryAB/HSPB2 germ line ablated (KO) and cardiac-specific CryAB overexpressingtransgenic (TG) mice. Pressure overload was induced by transverse aortic constriction in KO, TG, and non-transgenic wild type (NTG) control mice and 10 weeks later molecular, cellular, and whole organ level hypertrophic responses were analyzed. As we previously described, CryAB/HSPB2 KO mice showed abnormal baseline cardiac physiology that worsened into a restrictive cardiomyopathic phenotype with aging. Severe pressure overload in these mice led to rapid deterioration of heart function and development of congestive cardiac failure. Contrary to their short term protective phenotype, CryAB TG mice showed no significant effects on cardiac hypertrophic responses and very modest improvement of hemodynamics during chronic systolic overload. These findings indicate that small HSPs CryAB and/or HSPB2 are essential to maintain cardiac structure and function but overex-pression of CryAB is not sufficient to confer a sustained protection against chronic systolic overload. PMID:20733949

Protein quality control in protection against systolic overload cardiomyopathy: the long term role of small heat shock proteins.

PubMed

Kumarapeli, Asangi R K; Horak, Kathleen; Wang, Xuejun

2010-07-21

Molecular chaperones represent the first line of defense of intracellular protein quality control. As a major constituent of molecular chaperones, heat shock proteins (HSP) are known to confer cardiomyocyte short-term protection against various insults and injuries. Previously, we reported that the small HSP alphaB-crystallin (CryAB) attenuates cardiac hypertrophic response in mice subjected to 2 weeks of severe pressure overload. However, the long-term role of small HSPs in cardiac hypertrophy and failure has rarely been studied. The present study investigates the cardiac responses to chronic severe pressure overload in CryAB/HSPB2 germ line ablated (KO) and cardiac-specific CryAB overexpressingtransgenic (TG) mice. Pressure overload was induced by transverse aortic constriction in KO, TG, and non-transgenic wild type (NTG) control mice and 10 weeks later molecular, cellular, and whole organ level hypertrophic responses were analyzed. As we previously described, CryAB/HSPB2 KO mice showed abnormal baseline cardiac physiology that worsened into a restrictive cardiomyopathic phenotype with aging. Severe pressure overload in these mice led to rapid deterioration of heart function and development of congestive cardiac failure. Contrary to their short term protective phenotype, CryAB TG mice showed no significant effects on cardiac hypertrophic responses and very modest improvement of hemodynamics during chronic systolic overload. These findings indicate that small HSPs CryAB and/or HSPB2 are essential to maintain cardiac structure and function but overex-pression of CryAB is not sufficient to confer a sustained protection against chronic systolic overload.

Field-Evolved Resistance in Corn Earworm to Cry Proteins Expressed by Transgenic Sweet Corn

PubMed Central

Dively, Galen P.; Finkenbinder, Chad

2016-01-01

Background Transgenic corn engineered with genes expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Berliner) (Bt) are now a major tool in insect pest management. With its widespread use, insect resistance is a major threat to the sustainability of the Bt transgenic technology. For all Bt corn expressing Cry toxins, the high dose requirement for resistance management is not achieved for corn earworm, Helicoverpa zea (Boddie), which is more tolerant to the Bt toxins. Methodology/Major Findings We present field monitoring data using Cry1Ab (1996–2016) and Cry1A.105+Cry2Ab2 (2010–2016) expressing sweet corn hybrids as in-field screens to measure changes in field efficacy and Cry toxin susceptibility to H. zea. Larvae successfully damaged an increasing proportion of ears, consumed more kernel area, and reached later developmental stages (4th - 6th instars) in both types of Bt hybrids (Cry1Ab—event Bt11, and Cry1A.105+Cry2Ab2—event MON89034) since their commercial introduction. Yearly patterns of H. zea population abundance were unrelated to reductions in control efficacy. There was no evidence of field efficacy or tissue toxicity differences among different Cry1Ab hybrids that could contribute to the decline in control efficacy. Supportive data from laboratory bioassays demonstrate significant differences in weight gain and fitness characteristics between the Maryland H. zea strain and a susceptible strain. In bioassays with Cry1Ab expressing green leaf tissue, Maryland H. zea strain gained more weight than the susceptible strain at all concentrations tested. Fitness of the Maryland H. zea strain was significantly lower than that of the susceptible strain as indicated by lower hatch rate, longer time to adult eclosion, lower pupal weight, and reduced survival to adulthood. Conclusions/Significance After ruling out possible contributing factors, the rapid change in field efficacy in recent years and decreased susceptibility of H. zea to Bt sweet corn provide strong evidence of field-evolved resistance in H. zea populations to multiple Cry toxins. The high adoption rate of Bt field corn and cotton, along with the moderate dose expression of Cry1Ab and related Cry toxins in these crops, and decreasing refuge compliance probably contributed to the evolution of resistance. Our results have important implications for resistance monitoring, refuge requirements and other regulatory policies, cross-resistance issues, and the sustainability of the pyramided Bt technology. PMID:28036388

Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera.

PubMed

Singh, Shweta; Kumar, Nikhil Ram; Maniraj, R; Lakshmikanth, R; Rao, K Y S; Muralimohan, N; Arulprakash, T; Karthik, K; Shashibhushan, N B; Vinutha, T; Pattanayak, Debasis; Dash, Prasanta K; Kumar, P Ananda; Sreevathsa, Rohini

2018-06-11

Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T 1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T 3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80-100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25-80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.

Bitrophic and Tritrophic Effects of Transgenic cry1Ab/cry2Aj Maize on the Beneficial, Nontarget Harmonia axyridis (Coleoptera: Coccinellidae).

PubMed

Chang, Xue; Lu, Zengbin; Shen, Zhicheng; Peng, Yufa; Ye, Gongyin

2017-10-01

Harmonia axyridis (Pallas) is a common and abundant predator in China and may be exposed to Cry toxins that are produced in Bt crops either by feeding on plant parts or by feeding on target or nontarget herbivorous insects. A new Bt maize line, expressing the Cry1Ab/Cry2Aj fused protein, has been developed and should be rigorously assessed for the ecological risks on the natural enemy. Laboratory experiments were carried out to study the effects of this Bt maize on nontarget predator H. axyridis via bitrophic interaction of adult H. axyridis feeding on Bt maize pollen and tritrophic interaction of H. axyridis consuming the lepidopteran prey. Spodoptera exigua (Hübner) neonate larvae were used to transfer Bt protein because they could survive after ingesting transgenic cry1Ab/cry2Aj maize kernels in the previous study. ELISA bioassays confirmed that the Bt protein could be transferred, but diluted through Bt maize-prey-predator. Life history parameters such as survival, development, weight, fecundity, and egg hatching rate were not significantly different when H. axyridis consumed prey that had been reared on Bt maize compared with prey reared on a nontransformed parental control. Furthermore, feeding directly on Bt maize pollen also had no detrimental effects on fitness, survival, and weight of female and male adults. In conclusion, our results indicate that transgenic cry1Ab/cry2Aj maize poses no ecological risks on the nontarget predator H. axyridis. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Field-evolved resistance to Cry1Ab maize by Spodoptera frugiperda in Brazil.

PubMed

Omoto, Celso; Bernardi, Oderlei; Salmeron, Eloisa; Sorgatto, Rodrigo J; Dourado, Patrick M; Crivellari, Augusto; Carvalho, Renato A; Willse, Alan; Martinelli, Samuel; Head, Graham P

2016-09-01

The first Bt maize in Brazil was launched in 2008 and contained the MON 810 event, which expresses Cry1Ab protein. Although the Cry1Ab dose in MON 810 is not high against fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), MON 810 provided commercial levels of control. To support insect resistance management in Brazil, the baseline and ongoing susceptibility of FAW was examined using protein bioassays, and the level of control and life history parameters of FAW were evaluated on MON 810 maize. Baseline diet overlay assays with Cry1Ab (16 µg cm(-2) ) caused 76.3% mortality to field FAW populations sampled in 2009. Moderate mortality (48.8%) and significant growth inhibition (88.4%) were verified in leaf-disc bioassays. In greenhouse trials, MON 810 had significantly less damage than non-Bt maize. The surviving FAW larvae on MON 810 (22.4%) had a 5.5 day increase in life cycle time and a 24% reduction in population growth rate. Resistance monitoring (2010-2015) showed a significant reduction in Cry1Ab susceptibility of FAW over time. Additionally, a significant reduction in the field efficacy of MON 810 maize against FAW was observed in different regions from crop season 2009 to 2013. The decrease in susceptibility to Cry1Ab was expected, but the specific contributions to this resistance by MON 810 maize cannot be distinguished from cross-resistance to Cry1Ab caused by exposure to Cry1F maize. Technologies combining multiple novel insecticidal traits with no cross-resistance to the current Cry1 proteins and high activity against the same target pests should be pursued in Brazil and similar environments. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

A 52-week safety study in cynomolgus macaques for genetically modified rice expressing Cry1Ab/1Ac protein.

PubMed

Mao, Jie; Sun, Xing; Cheng, Jian-Hua; Shi, Yong-Jie; Wang, Xin-Zheng; Qin, Jun-Jie; Sang, Zhi-Hong; He, Kun; Xia, Qing

2016-09-01

A 52-week feeding study in cynomolgus macaques was carried out to evaluate the safety of Bt rice Huahui 1 (HH1), a transgenic rice line expressing Cry1Ab/1Ac protein. Monkeys were fed a diet with 20% or 60% HH1 rice, 20% or 60% parental rice (Minghui 63, MH63), normal diet, normal diet spiked with purified recombinant Cry1Ab/1Ac fusion protein or bovine serum albumin (BSA) respectively. During the feeding trail, clinical observations were conducted daily, and multiple parameters, including body weight, body temperature, electrocardiogram, hematology, blood biochemistry, serum metabolome and gut microbiome were examined at regular intervals. Upon sacrifice, the organs were weighted, and the macroscopic, microscopic and electron microscopic examinations were performed. The results show no adverse or toxic effects of Bt rice HH1 or Cry1Ab/1Ac fusion protein on monkeys. Therefore, the present 52-week primate feeding study suggests that the transgenic rice containing Cry 1Ab/1Ac is equivalent to its parental rice line MH63. Copyright © 2016 Elsevier Ltd. All rights reserved.

LRIG2 mutations cause urofacial syndrome.

PubMed

Stuart, Helen M; Roberts, Neil A; Burgu, Berk; Daly, Sarah B; Urquhart, Jill E; Bhaskar, Sanjeev; Dickerson, Jonathan E; Mermerkaya, Murat; Silay, Mesrur Selcuk; Lewis, Malcolm A; Olondriz, M Beatriz Orive; Gener, Blanca; Beetz, Christian; Varga, Rita E; Gülpınar, Omer; Süer, Evren; Soygür, Tarkan; Ozçakar, Zeynep B; Yalçınkaya, Fatoş; Kavaz, Aslı; Bulum, Burcu; Gücük, Adnan; Yue, Wyatt W; Erdogan, Firat; Berry, Andrew; Hanley, Neil A; McKenzie, Edward A; Hilton, Emma N; Woolf, Adrian S; Newman, William G

2013-02-07

Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease. We have previously shown that UFS is also caused by mutations in HPSE2, encoding heparanase-2. LRIG2 and heparanase-2 were immunodetected in nerve fascicles growing between muscle bundles within the human fetal bladder, directly implicating both molecules in neural development in the lower urinary tract. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The food and environmental safety of Bt crops.

PubMed

Koch, Michael S; Ward, Jason M; Levine, Steven L; Baum, James A; Vicini, John L; Hammond, Bruce G

2015-01-01

Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms.

The food and environmental safety of Bt crops

PubMed Central

Koch, Michael S.; Ward, Jason M.; Levine, Steven L.; Baum, James A.; Vicini, John L.; Hammond, Bruce G.

2015-01-01

Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms. PMID:25972882

Establishment of Cry9C susceptibility baselines for European corn borer and southwestern corn borer (Lepidoptera: Crambidae).

PubMed

Reed, J P; Halliday, W R

2001-04-01

In 1997 and 1998, Cry9C susceptibility baselines were established for field-collected populations of European corn borer, Osrinia nubilalis (Hubner), and southwestern corn borer, Diatraea grandiosella Dyar. Bioassay of neonate European corn borer larvae of 16 colonies collected from the midwestern United States indicated LC50 values ranging from 13.2 to 65.1 ng of Cry9C protein per square centimeter. Neonate European corn borer LC50 values ranged from 46.5 to 214 ng/cm2. Neonate larvae of three colonies of southwestern corn borer collected from the southern and southwestern United States exhibited LC50 values from 16.9 to 39.9 ng of Cry9C protein per square centimeter. Southwestern corn borer neonate LC90 confidence limit values ranged from 40.3 to 157 ng of Cry9C protein per centimeter. The most sensitive southwestern corn borer colony was collected from the Mississippi delta exhibiting an LC50 value of 22.6 ng of Cry9C per cm2 and also displayed the widest LC0 confidence limits of 40.3-94.8 ng of Cry9C per cm2. Geographic baseline susceptibility data establishes the natural genetic variation and provides the foundation for future testing of insect populations exposed to increased use of Bacillus thuringiensis-based crops. Insect resistance management and stewardship of Cry9C will rely upon baseline data for the validation of discriminating dose assays for European corn borer and southwestern corn borer.

Changes in growth conditions alter the male strobilus gene expression pattern in Cryptomeria japonica.

PubMed

Fukui, Mitsue

2003-11-01

Two-year old saplings grown from cuttings of Cryptomeria japonica D. Don initiate strobilus development following treatment with gibberellic acid under long-day photoperiods. At 25 degrees C with a 14-h photoperiod in a phytotron, male strobili initiated normally; however, they remained green and fell from the saplings prematurely. To examine the change in male strobilus development at the molecular level, three genes expressed specifically in male strobili were analyzed. Two were MADS box genes homologous to the B-function genes in angiosperms, CjMADS1 and CjMADS2, and the third was Cry j I, which encodes an allergen protein, and this gene is expressed mainly in microspores. Under phytotron growing conditions, the homeotic genes were expressed constantly, which reflected the extended early developmental stage of male strobili. On the other hand, Cry j I expression was detected after a long delay just before strobilus development ceased. These results indicate that the expression of the genes related to male reproductive development in C. japonica is regulated by a factor(s) that is sensitive to environmental signals.

Multi-Toxin Resistance Enables Pink Bollworm Survival on Pyramided Bt Cotton

PubMed Central

Fabrick, Jeffrey A.; Unnithan, Gopalan C.; Yelich, Alex J.; DeGain, Ben; Masson, Luke; Zhang, Jie; Carrière, Yves; Tabashnik, Bruce E.

2015-01-01

Transgenic crops producing Bacillus thuringiensis (Bt) proteins kill key insect pests, providing economic and environmental benefits. However, the evolution of pest resistance threatens the continued success of such Bt crops. To delay or counter resistance, transgenic plant “pyramids” producing two or more Bt proteins that kill the same pest have been adopted extensively. Field populations of the pink bollworm (Pectinophora gossypiella) in the United States have remained susceptible to Bt toxins Cry1Ac and Cry2Ab, but field-evolved practical resistance to Bt cotton producing Cry1Ac has occurred widely in India. Here we used two rounds of laboratory selection to achieve 18,000- to 150,000-fold resistance to Cry2Ab in pink bollworm. Inheritance of resistance to Cry2Ab was recessive, autosomal, conferred primarily by one locus, and independent of Cry1Ac resistance. We created a strain with high resistance to both toxins by crossing the Cry2Ab-resistant strain with a Cry1Ac-resistant strain, followed by one selection with Cry2Ab. This multi-toxin resistant strain survived on field-collected Bt cotton bolls producing both toxins. The results here demonstrate the risk of evolution of resistance to pyramided Bt plants, particularly when toxins are deployed sequentially and refuges are scarce, as seen with Bt cotton and pink bollworm in India. PMID:26559899

Intra-vitreal αB crystallin fused to elastin-like polypeptide provides neuroprotection in a mouse model of age-related macular degeneration.

PubMed

Sreekumar, Parameswaran G; Li, Zhe; Wang, Wan; Spee, Christine; Hinton, David R; Kannan, Ram; MacKay, J Andrew

2018-05-18

Age-related macular degeneration (AMD) is the leading cause of severe and irreversible central vision loss, and the primary site of AMD pathology is the retinal pigment epithelium (RPE). Geographic atrophy (GA) is an advanced form of AMD characterized by extensive RPE cell loss, subsequent degeneration of photoreceptors, and thinning of retina. This report describes the protective potential of a peptide derived from the αB crystallin protein using a sodium iodate (NaIO 3 ) induced mouse model of GA. Systemic NaIO 3 challenge causes degeneration of the RPE and neighboring photoreceptors, which have similarities to retinas of GA patients. αB crystallin is an abundant ocular protein that maintains ocular clarity and retinal homeostasis, and a small peptide from this protein (mini cry) displays neuroprotective properties. To retain this peptide for longer in the vitreous, mini cry was fused to an elastin-like polypeptide (ELP). A single intra-vitreal treatment by this crySI fusion significantly inhibits retinal degeneration in comparison to free mini cry. While mini cry is cleared from the eye with a mean residence time of 0.4 days, crySI is retained with a mean residence time of 3.0 days; furthermore, fundus photography reveals evidence of retention at two weeks. Unlike the free mini cry, crySI protects the RPE against NaIO 3 challenge for at least two weeks after administration. CrySI also inhibits RPE apoptosis and caspase-3 activation and protects the retina from cell death up to 1-month post NaIO 3 challenge. These results show that intra-ocular ELP-linked peptides such as crySI hold promise as protective agents to prevent RPE atrophy and progressive retinal degeneration in AMD. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of seed blends and soil-insecticide on western and northern corn rootworm emergence from mCry3A + eCry3.1Ab Bt maize

USDA-ARS?s Scientific Manuscript database

Blended seed mixtures containing various ratios of transgenic Bt maize expressing the mCry3A + eCry3.1Ab proteins and non-Bt maize (near-isoline maize) were deployed alone and in combination with a soil applied insecticide (Force CS) to evaluate the survivorship of the western corn rootworm, Diabrot...

Short-Term Evaluation in Growing Rats of Diet Containing Bacillus thuringiensis Cry1Ia12 Entomotoxin: Nutritional Responses and Some Safety Aspects

PubMed Central

Guimarães, Luciane Mourão; Farias, Davi Felipe; Muchagata, Relinda Campos Carvalho; de Magalhães, Mariana Quezado; Campello, Cláudio Cabral; Rocha, Thales Lima; Vasconcelos, Ilka Maria; Carvalho, Ana Fontenele Urano; Mulinari, Fernanda; Grossi-de-Sa, Maria Fátima

2010-01-01

The Cry1Ia12 entomotoxin from a Brazilian Bacillus thuringiensis strain is currently being expressed in cotton cultivars to confer resistance to insect-pests. The present study aimed to assess the effects of a diet containing Cry1Ia12 protein on growing rats. A test diet containing egg white and Cry1Ia12 (0.1% of total protein) as a protein source was offered to rats for ten days. In addition, an acute toxicity bioassay was performed in rats with a single oral dose of the entomotoxin (12 mg/animal). No adverse effects were observed in the animals receiving the test diet when compared to those receiving a control diet (egg white). The analysed parameters included relative dry weight of internal organs, duodenum histology, blood biochemistry, and nutritional parameters. The results of the acute toxicity test showed no mortality or behaviour alteration. Thus, Cry1Ia12 toxin at the tested concentration does not cause deleterious effects on growing rats when incorporated in the diet for 10 days. PMID:20862341

Characterization of the Activity Spectrum of MON 88702 and the Plant-Incorporated Protectant Cry51Aa2.834_16

PubMed Central

Ahmad, Aqeel; Ahrens, Jeffrey E.; Akbar, Waseem; Baum, James A.; Brown, Scott; Clark, Thomas L.; Fridley, Jennifer M.; Gowda, Anilkumar; Greenplate, John T.; Jensen, Peter D.; Mueller, Geoffrey M.; Odegaard, Matthew L.; Tan, Jianguo; Uffman, Joshua P.; Levine, Steven L.

2017-01-01

The spectrum of insecticidal activity of Cry51Aa2.834_16 protein targeting hemipteran and thysanopteran insect pests in cotton was characterized by selecting and screening multiple pest and non-pest species, based on representation of ecological functional groups, taxonomic relatedness (e.g. relationship to species where activity was observed), and availability for effective testing. Seven invertebrate orders, comprising 12 families and 17 representative species were screened for susceptibility to Cry51Aa2.834_16 protein and/or the ability of the protein to protect against feeding damage in laboratory, controlled environments (e.g. greenhouse/growth chamber), and/or field studies when present in cotton plants. The screening results presented for Cry51Aa2.834_16 demonstrate selective and limited activity within three insect orders. Other than Orius insidiosus, no activity was observed for Cry51Aa2.834_16 against several groups of arthropods that perform key ecological roles in some agricultural ecosystems (e.g. pollinators, decomposers, and natural enemies). PMID:28072875

Unilateral incompatibility gene ui1.1 encodes an S-locus F-box protein expressed in pollen of Solanum species.

PubMed

Li, Wentao; Chetelat, Roger T

2015-04-07

Unilateral interspecific incompatibility (UI) is a postpollination, prezygotic reproductive barrier that prevents hybridization between related species when the female parent is self-incompatible (SI) and the male parent is self-compatible (SC). In tomato and related Solanum species, two genes, ui1.1 and ui6.1, are required for pollen compatibility on pistils of SI species or hybrids. We previously showed that ui6.1 encodes a Cullin1 (CUL1) protein. Here we report that ui1.1 encodes an S-locus F-box (SLF) protein. The ui1.1 gene was mapped to a 0.43-cM, 43.2-Mbp interval at the S-locus on chromosome 1, but positional cloning was hampered by low recombination frequency. We hypothesized that ui1.1 encodes an SLF protein(s) that interacts with CUL1 and Skp1 proteins to form an SCF-type (Skp1, Cullin1, F-box) ubiquitin E3 ligase complex. We identified 23 SLF genes in the S. pennellii genome, of which 19 were also represented in cultivated tomato (S. lycopersicum). Data from recombination events, expression analysis, and sequence annotation highlighted 11 S. pennellii genes as candidates. Genetic transformations demonstrated that one of these, SpSLF-23, is sufficient for ui1.1 function. A survey of cultivated and wild tomato species identified SLF-23 orthologs in each of the SI species, but not in the SC species S. lycopersicum, S. cheesmaniae, and S. galapagense, pollen of which lacks ui1.1 function. These results demonstrate that pollen compatibility in UI is mediated by protein degradation through the ubiquitin-proteasome pathway, a mechanism related to that which controls pollen recognition in SI.

Concentrated Protein Body Product Derived from Rice Endosperm as an Oral Tolerogen for Allergen-Specific Immunotherapy—A New Mucosal Vaccine Formulation against Japanese Cedar Pollen Allergy

PubMed Central

Wakasa, Yuhya; Takagi, Hidenori; Watanabe, Nobumasa; Kitamura, Noriko; Fujiwara, Yoshihiro; Ogo, Yuko; Hayashi, Shimpei; Yang, Lijun; Ohta, Masaru; Thet Tin, Wai Wai; Sekikawa, Kenji; Takano, Makoto; Ozawa, Kenjirou; Hiroi, Takachika; Takaiwa, Fumio

2015-01-01

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation. PMID:25774686

Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae

USDA-ARS?s Scientific Manuscript database

Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and th...

A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides.

PubMed

Hendrischk, Anne-Kathrin; Frühwirth, Sebastian Walter; Moldt, Julia; Pokorny, Richard; Metz, Sebastian; Kaiser, Gebhard; Jäger, Andreas; Batschauer, Alfred; Klug, Gabriele

2009-11-01

Blue light receptors belonging to the cryptochrome/photolyase family are found in all kingdoms of life. The functions of photolyases in repair of UV-damaged DNA as well as of cryptochromes in the light-dependent regulation of photomorphogenetic processes and in the circadian clock in plants and animals are well analysed. In prokaryotes, the only role of members of this protein family that could be demonstrated is DNA repair. Recently, we identified a gene for a cryptochrome-like protein (CryB) in the alpha-proteobacterium Rhodobacter sphaeroides. The protein lacks the typical C-terminal extension of cryptochromes, and is not related to the Cry DASH family. Here we demonstrate that CryB binds flavin adenine dinucleotide that can be photoreduced by blue light. CryB binds single-stranded DNA with very high affinity (K(d) approximately 10(-8) M) but double-stranded DNA and single-stranded RNA with far lower affinity (K(d) approximately 10(-6) M). Despite of that, no in vitro repair activity for pyrimidine dimers in single-stranded DNA could be detected. However, we show that CryB clearly affects the expression of genes for pigment-binding proteins and consequently the amount of photosynthetic complexes in R. sphaeroides. Thus, for the first time a role of a bacterial cryptochrome in gene regulation together with a biological function is demonstrated.

What are farmers really planting? Measuring the presence and effectiveness of Bt cotton in Pakistan.

PubMed

Spielman, David J; Zaidi, Fatima; Zambrano, Patricia; Khan, Asif Ali; Ali, Shaukat; Cheema, H Masooma Naseer; Nazli, Hina; Khan, Rao Sohail Ahmad; Iqbal, Arshad; Zia, Muhammad Amir; Ali, Ghulam Muhammad

2017-01-01

Genetically modified, insect-resistant Bacillus thuringiensis (Bt) cotton is cultivated extensively in Pakistan. Past studies, however, have raised concerns about the prevalence of Bt cotton varieties possessing weak or nonperforming insect-resistance traits conferred by the cry gene. We examine this issue using data drawn from a representative sample of cotton-growing households that were surveyed in six agroclimatic zones spanning 28 districts in Pakistan in 2013, as well as measurements of Cry protein levels in cotton tissue samples collected from the sampled households' main fields. The resultant dataset combines information from 593 sampled households with corresponding plant tissue diagnostics from 70 days after sowing, as well as information from 589 sampled households with corresponding diagnostics from 120 days after sowing. Our analysis indicates that 11 percent of farmers believed they were cultivating Bt cotton when, in fact, the Cry toxin was not present in the tested tissue at 70 days after sowing (i.e., a Type I error). The analysis further indicates that 5 percent of farmers believed they were cultivating non-Bt cotton when, in fact, the Cry toxin was present in the tested tissue (i.e., a Type II error). In addition, 17 percent of all sampled farmers were uncertain whether or not they were cultivating Bt cotton. Overall, 33 percent of farmers either did not know or were mistaken in their beliefs about the presence of the cry gene in the cotton they cultivated. Results also indicate that toxic protein levels in the plant tissue samples occurred below threshold levels for lethality in a significant percentage of cases, although these measurements may also be affected by factors related to tissue sample collection, handling, storage, and testing procedures. Nonetheless, results strongly suggest wide variability both in farmers' beliefs and in gene expression. Such variability has implications for policy and regulation in Pakistan's transgenic cotton seed market.

Soil incubation studies with Cry1Ac protein indicate no adverse effect of Bt crops on soil microbial communities.

PubMed

Zhaolei, Li; Naishun, Bu; Xueping, Chen; Jun, Cui; Manqiu, Xiao; Zhiping, Song; Ming, Nie; Changming, Fang

2018-05-15

Bt crops that are transgenic crops engineered to produce Bt toxins which occur naturally with Bacillus thuringiensis (Bt) have been widely planted and its environmental risk assessment has been heavily debated. The effects of Bt crops on soil microbial communities are possible through changing the quantity and quality of C inputs and potential toxic activity of Bt protein on soil organisms. To date, the direct effects of Bt protein on soil microorganisms is unclear. Here we added Cry1Ac, one of the most commonly used Bt protein in Bt crops, to the soil and monitored changes in soil bacterial, fungal and archaeal diversities and community structures using ribosomal DNA-fingerprinting method, as well as their population sizes by real-time PCR over a 100-day period. Despite the fact that variations were observed in the indices of evenness, diversity and population sizes of bacteria, fungi and archaea with different Cry1Ac addition rates up to 100ngg -1 soil, the indices of soil microbial diversities and evennesses did not significantly shift with Cry1Ac protein addition, nor did population sizes change over time. The diversities of the dominant bacteria, fungi and archaea were not significantly changed, given Cry1Ac protein addition rates over a period of 100 days. These results suggested that Bt protein derived by cultivations of transgenic Bt crops is unlikely to cause transient or even persisting significant changes in soil microorganisms in field. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of hybrid fusion protein (Cry1Ac::ASAL) in transgenic rice plants imparts resistance against multiple insect pests.

PubMed

Boddupally, Dayakar; Tamirisa, Srinath; Gundra, Sivakrishna Rao; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

2018-05-31

To evolve rice varieties resistant to different groups of insect pests a fusion gene, comprising DI and DII domains of Bt Cry1Ac and carbohydrate binding domain of garlic lectin (ASAL), was constructed. Transgenic rice lines were generated and evaluated to assess the efficacy of Cry1Ac::ASAL fusion protein against three major pests, viz., yellow stem borer (YSB), leaf folder (LF) and brown planthopper (BPH). Molecular analyses of transgenic plants revealed stable integration and expression of the fusion gene. In planta insect bioassays on transgenics disclosed enhanced levels of resistance compared to the control plants. High insect mortality of YSB, LF and BPH was observed on transgenics compared to that of control plants. Furthermore, honeydew assays revealed significant decreases in the feeding ability of BPH on transgenic plants as compared to the controls. Ligand blot analysis, using BPH insects fed on cry1Ac::asal transgenic rice plants, revealed a modified receptor protein-binding pattern owing to its ability to bind to additional receptors in insects. The overall results authenticate that Cry1Ac::ASAL protein is endowed with remarkable entomotoxic effects against major lepidopteran and hemipteran insects. As such, the fusion gene appears promising and can be introduced into various other crops to control multiple insect pests.

A review of Cry protein detection with enzyme-linked immunosorbent assays

USDA-ARS?s Scientific Manuscript database

Several detection methods are available to monitor the fate of Cry proteins in the environment, enzyme-linked immunosorbent assays (ELISAs) have emerged as the preferred detection method, due to their cost-effectiveness, ease of use, and rapid results. Validation of ELISAs is necessary to ensure acc...

Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab

PubMed Central

Tabashnik, Bruce E.; Fabrick, Jeffrey A.; Unnithan, Gopalan C.; Yelich, Alex J.; Masson, Luke; Zhang, Jie; Bravo, Alejandra; Soberón, Mario

2013-01-01

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both. PMID:24244692

An ultrasensitive label-free electrochemiluminescent immunosensor for measuring Cry1Ab level and genetically modified crops content.

PubMed

Gao, Hongfei; Wen, Luke; Wu, Yuhua; Fu, Zhifeng; Wu, Gang

2017-11-15

The development of genetically modified (GM) insect-resistant crops has aroused great public concern about the risks on the eco-environment resulting from a release of toxic Cry proteins (such as Cry1Ab) to the soil. Therefore, it is of crucial importance to measure the Cry proteins level and the GM crops content. Here, we have tested for the first time a method that uses novel carbon nanospheres (CNPs) label-free electrochemiluminescent (ECL) immunosensor for the ultrasensitive quantification of Cry1Ab and GM crops. In this work, novel CNPs were prepared from printer toner with a very facile approach, and linked with anti-Cry1Ab antibodies to modify a golden working electrode. The immunoreaction between Cry1Ab and its antibody formed an immunocomplex on the bioreceptor region of the sensor, which inhibited electron transfer between the electrode surface and the ECL substance, leading to a decrease of ECL response. Under the optimal conditions, the fabricated label-free ECL immunosensor determined Cry1Ab down to 3.0pgmL -1 within a linear range of 0.010-1.0ngmL -1 , showing significant improvement of sensitivity than that of most previous reports. Meanwhile, the proposed method was successfully applied for GM rice BT63 and GM maize MON810 detections down to 0.010% and 0.020%, respectively. Due to its outstanding advantages such as high sensitivity, ideal selectivity, simple fabrication, rapid detection, and low cost, the developed method can be considered as a powerful and pioneering tool for GM crops detection. Its use can also be extended to other toxin protein sensing in foods. Copyright © 2017. Published by Elsevier B.V.

A specific binding protein from Tenebrio molitor for the insecticidal toxin of Bacillus thuringiensis subsp. tenebrionis.

PubMed

Belfiore, C J; Vadlamudi, R K; Osman, Y A; Bulla, L A

1994-04-15

Biopesticides based on the bacterium Bacillus thuringiensis have attracted wide attention as safe alternatives to chemical insecticides. In this paper, we report, for the first time, the identification of a single binding protein from a coleopteran insect, Tenebrio molitor, that is specific for the cryIII toxin of B. thuringiensis. The protein appeared as a single band of 144 kDa on radioligand and immunoblots of total proteins extracted from brush border membrane vesicles of the midgut of T. molitor. Radiolabelled cryIIIA toxin bound to the protein with a Kd value of 17.5 nM and could be specifically blocked by unlabelled toxin but not by toxins from other subspecies of B. thuringiensis. This study lays the groundwork to clone the cryIIIA toxin binding protein and to determine the molecular mechanism(s) of toxin action.

F2 screen for resistance to Bacillus thuringiensis Cry2Ab2-maize in field populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) from the southern United States.

PubMed

Niu, Ying; Qureshi, Jawwad A; Ni, Xinzhi; Head, Graham P; Price, Paula A; Meagher, Robert L; Kerns, David; Levy, Ronnie; Yang, Xiangbing; Huang, Fangneng

2016-07-01

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target pest of transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins in both North and South America. In 2013 and 2014, a total of 215 F2 two-parent families of S. frugiperda were established using single-pair mating of field individuals collected from seven locations in four states of the southern U.S.: Texas, Louisiana, Georgia, and Florida. The objective of the investigation was to detect resistance alleles in field populations to Cry2Ab2, a common Bt protein produced in transgenic maize and cotton. For each F2 family, 128 F2 neonates were screened on leaf tissue of Cry2Ab2 maize plants in the laboratory. A conservative estimate of the frequency of major Cry2Ab2 resistance alleles in S. frugiperda from the four states was 0.0023 with a 95% credibility interval of 0.0003-0.0064. In addition, six families were considered to likely possess minor resistance alleles at a frequency of 0.0082 with a 95% credibility interval of 0.0033-0.0152. One F2 family from Georgia (GA-15) was confirmed to possess a major resistance allele to the Cry2Ab2 protein. Larvae from this family survived well on whole maize plants expressing Cry2Ab2 protein and demonstrated a significant level (>15-fold) of resistance when fed with the same protein incorporated in a meridic diet. The detection of the major resistance allele along with the relatively abundant minor resistance alleles revealed in this study may have important implications for resistance management. Copyright © 2016 Elsevier Inc. All rights reserved.

Domain III of Cry1Ac Is Critical to Binding and Toxicity against Soybean Looper (Chrysodeixis includens) but Not to Velvetbean Caterpillar (Anticarsia gemmatalis).

PubMed

Mushtaq, Rubina; Shakoori, Abdul Rauf; Jurat-Fuentes, Juan Luis

2018-02-27

Insecticidal proteins Cry1Ac and Cry2Ac7 from the bacterium Bacillus thuringiensis (Bt) belong to the three-domain family of Bt toxins. Commercial transgenic soybean hybrids produce Cry1Ac to control the larvae of the soybean looper ( Chrysodeixis includens ) and the velvet bean caterpillar ( Anticarsia gemmatalis ). The specificity of Cry1Ac is determined by loops extending from domain II and regions of domain III in the three-dimensional structure of the toxin. In this study, we constructed a hybrid toxin (H1.2Ac) containing domains I and II of Cry1Ac and domain III of Cry2Ac7, in an attempt to obtain a protein with enhanced toxicity compared to parental toxins. Bioassays with H1.2Ac revealed toxicity against the larvae of A. gemmatalis but not against C. includens . Saturation binding assays with radiolabeled toxins and midgut brush border membrane vesicles demonstrated no specific H1.2Ac binding to C. includens , while binding in A. gemmatalis was specific and saturable. Results from competition binding assays supported the finding that Cry1Ac specificity against A. gemmatalis is mainly dictated by domain II. Taken together, these distinct interactions with binding sites may help explain the differential susceptibility to Cry1Ac in C. includens and A. gemmatalis , and guide the design of improved toxins against soybean pests.

Analysis of cross-resistance to Vip3 proteins in eight insect colonies selected for resistance to Bacillus thuringiensis insecticidal proteins

USDA-ARS?s Scientific Manuscript database

Bacillus thuringiensis Vip3 proteins are synthesized and secreted during the vegetative growth phase. They are activated by gut proteases, recognize and bind to midgut receptors, form pores and lyse cells. We tested the susceptibility to Vip3Aa and Vip3Ca of Cry1A-, Cry2A-, Dipel- and Vip3-resistant...

Cry3Bb1-Resistant Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte) Does Not Exhibit Cross-Resistance to DvSnf7 dsRNA.

PubMed

Moar, William; Khajuria, Chitvan; Pleau, Michael; Ilagan, Oliver; Chen, Mao; Jiang, Changjian; Price, Paula; McNulty, Brian; Clark, Thomas; Head, Graham

2017-01-01

There is a continuing need to express new insect control compounds in transgenic maize against western corn rootworm, Diabrotica virgifera virgifera (LeConte) (WCR). In this study three experiments were conducted to determine cross-resistance between the new insecticidal DvSnf7 dsRNA, and Bacillus thuringiensis (Bt) Cry3Bb1; used to control WCR since 2003, with field-evolved resistance being reported. Laboratory susceptible and Cry3Bb1-resistant WCR were evaluated against DvSnf7 dsRNA in larval diet-incorporation bioassays. Additionally, the susceptibility of seven field and one field-derived WCR populations to DvSnf7 (and Cry3Bb1) was assessed in larval diet-overlay bioassays. Finally, beetle emergence of laboratory susceptible and Cry3Bb1-resistant WCR was evaluated with maize plants in the greenhouse expressing Cry3Bb1, Cry34Ab1/Cry35Ab1, or DvSnf7 dsRNA singly, or in combination. The Cry3Bb1-resistant colony had slight but significantly (2.7-fold; P<0.05) decreased susceptibility to DvSnf7 compared to the susceptible colony, but when repeated using a field-derived WCR population selected for reduced Cry3Bb1 susceptibility, there was no significant difference (P<0.05) in DvSnf7 susceptibility compared to that same susceptible population. Additionally, this 2.7-fold difference in susceptibility falls within the range of DvSnf7 susceptibility among the seven field populations tested. Additionally, there was no correlation between susceptibility to DvSnf7 and Cry3Bb1 for all populations evaluated. In greenhouse studies, there were no significant differences (P<0.05) between beetle emergence of susceptible and Cry3Bb1-resistant colonies on DvSnf7 and Cry34Ab1/Cry35Ab1, and between DvSnf7 and MON 87411 (DvSnf7 + Cry3Bb1) for the Cry3Bb1-resistant colony. These results demonstrate no cross-resistance between DvSnf7 and Cry3Bb1 against WCR. Therefore, pyramiding DvSnf7 with Bt proteins such as Cry3Bb1 and Cry34Ab1/Cry35Ab1 will provide a valuable IRM tool against WCR that will increase the durability of these Bt proteins. These results also illustrate the importance of using appropriate bioassay methods when characterizing field-evolved resistant WCR populations.

Cry3Bb1-Resistant Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte) Does Not Exhibit Cross-Resistance to DvSnf7 dsRNA

PubMed Central

Khajuria, Chitvan; Pleau, Michael; Ilagan, Oliver; Chen, Mao; Jiang, Changjian; Price, Paula; McNulty, Brian; Clark, Thomas; Head, Graham

2017-01-01

Background and Methodology There is a continuing need to express new insect control compounds in transgenic maize against western corn rootworm, Diabrotica virgifera virgifera (LeConte) (WCR). In this study three experiments were conducted to determine cross-resistance between the new insecticidal DvSnf7 dsRNA, and Bacillus thuringiensis (Bt) Cry3Bb1; used to control WCR since 2003, with field-evolved resistance being reported. Laboratory susceptible and Cry3Bb1-resistant WCR were evaluated against DvSnf7 dsRNA in larval diet-incorporation bioassays. Additionally, the susceptibility of seven field and one field-derived WCR populations to DvSnf7 (and Cry3Bb1) was assessed in larval diet-overlay bioassays. Finally, beetle emergence of laboratory susceptible and Cry3Bb1-resistant WCR was evaluated with maize plants in the greenhouse expressing Cry3Bb1, Cry34Ab1/Cry35Ab1, or DvSnf7 dsRNA singly, or in combination. Principal Findings and Conclusions The Cry3Bb1-resistant colony had slight but significantly (2.7-fold; P<0.05) decreased susceptibility to DvSnf7 compared to the susceptible colony, but when repeated using a field-derived WCR population selected for reduced Cry3Bb1 susceptibility, there was no significant difference (P<0.05) in DvSnf7 susceptibility compared to that same susceptible population. Additionally, this 2.7-fold difference in susceptibility falls within the range of DvSnf7 susceptibility among the seven field populations tested. Additionally, there was no correlation between susceptibility to DvSnf7 and Cry3Bb1 for all populations evaluated. In greenhouse studies, there were no significant differences (P<0.05) between beetle emergence of susceptible and Cry3Bb1-resistant colonies on DvSnf7 and Cry34Ab1/Cry35Ab1, and between DvSnf7 and MON 87411 (DvSnf7 + Cry3Bb1) for the Cry3Bb1-resistant colony. These results demonstrate no cross-resistance between DvSnf7 and Cry3Bb1 against WCR. Therefore, pyramiding DvSnf7 with Bt proteins such as Cry3Bb1 and Cry34Ab1/Cry35Ab1 will provide a valuable IRM tool against WCR that will increase the durability of these Bt proteins. These results also illustrate the importance of using appropriate bioassay methods when characterizing field-evolved resistant WCR populations. PMID:28060922

Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis).

PubMed

Oliveira, Gustavo R; Silva, Maria C M; Lucena, Wagner A; Nakasu, Erich Y T; Firmino, Alexandre A P; Beneventi, Magda A; Souza, Djair S L; Gomes, José E; de Souza, José D A; Rigden, Daniel J; Ramos, Hudson B; Soccol, Carlos R; Grossi-de-Sa, Maria F

2011-09-09

The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. In vitro directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of Bacillus thuringiensis. Bioassays carried out with A. grandis larvae revealed that the LC50 of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability. The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control A. grandis.

Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis)

PubMed Central

2011-01-01

Background The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. In vitro directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of Bacillus thuringiensis. Results Bioassays carried out with A. grandis larvae revealed that the LC50 of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability. Conclusions The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control A. grandis. PMID:21906288

A Role for Barley CRYPTOCHROME1 in Light Regulation of Grain Dormancy and Germination[W][OPEN

PubMed Central

Barrero, Jose M.; Downie, A. Bruce; Xu, Qian; Gubler, Frank

2014-01-01

It is well known that abscisic acid (ABA) plays a central role in the regulation of seed dormancy and that transcriptional regulation of genes encoding ABA biosynthetic and degradation enzymes is responsible for determining ABA content. However, little is known about the upstream signaling pathways impinging on transcription to ultimately regulate ABA content or how environmental signals (e.g., light and cold) might direct such expression in grains. Our previous studies indicated that light is a key environmental signal inhibiting germination in dormant grains of barley (Hordeum vulgare), wheat (Triticum aestivum), and Brachypodium distachyon and that this effect attenuates as after-ripening progresses further. We found that the blue component of the light spectrum inhibits completion of germination in barley by inducing the expression of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase and dampening expression of ABA 8’-hydroxylase, thus increasing ABA content in the grain. We have now created barley transgenic lines downregulating the genes encoding the blue light receptors CRYTOCHROME (CRY1) and CRY2. Our results demonstrate that CRY1 is the key receptor perceiving and transducing the blue light signal in dormant grains. PMID:24642944

Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana.

PubMed

Zirak, P; Penzkofer, A; Moldt, J; Pokorny, R; Batschauer, A; Essen, L-O

2009-11-09

The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FAD(ox)), semiquinone form (FADH(.)), and anionic hydroquinone form (FAD(red)H(-)). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FAD(ox) to the anionic semiquinone form (FAD()(-)) with a quantum efficiency of about 2% and a back recovery time of about 10s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FAD(ox) to FADH(.), FADH(.) to FAD(red)H(-), and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.

Use of JH4 joining segment gene by an anti-arsonate antibody that bears the major A-strain cross-reactive idiotype but displays diminished antigen binding.

PubMed

Slaughter, C A; Jeske, D J; Kuziel, W A; Milner, E C; Capra, J D

1984-06-01

One of the antibody families utilized by the A/J mouse in its response to p-azophenylarsonate (Ars) is characterized by the expression of the major anti-arsonate cross-reactive idiotype (CRI) of the A strain. This family has been termed the Ars-A family. A hybridoma antibody (HP 101F11 ) obtained after immunization of an A/J mouse with Ars was identified initially as displaying the CRI, but was subsequently found to bind antigen at a level much lower than most members of the Ars-A family. The results of binding studies suggested that HP 101F11 possesses reduced avidity for antigen. When isolated light and heavy chains were allowed to recombine with the heavy and light chains of a strongly antigen-binding, strongly CRI-positive antibody of the Ars-A family (HP 93G7 ), the low level of antigen binding by HP 101F11 was found to be due to a structurally variant heavy chain. Whereas antibodies of the Ars-A family with normal avidity for antigen had been shown to use the JH2 joining segment gene, amino acid sequence analysis of HP 101F11 revealed that this antibody has a JH segment with a sequence identical to that encoded by a portion of a different JH gene, JH4 . The implication that 101F11 uses the JH4 gene instead of JH2 was supported by the observation that the productively rearranged gene is associated with an Eco R1 restriction fragment 0.95 Kb smaller than the corresponding fragments of Ars-A hybridomas with normal avidity for antigen. The size difference of 0.95 Kb corresponds exactly to the known distance between the JH2 and JH4 genes in BALB/c germline DNA. In addition to the structural differences immediately attributable to the use of JH4 , HP 101F11 has shown an amino acid interchange in the DH segment, and a single amino acid deletion at the DH-JH boundary. These results show that variation among members of the Ars-A family in the DH and/or JH segments provides alternative structural forms of Ars-A antibodies upon which selective processes can operate during the course of an immune response.

Residues at a Single Site Differentiate Animal Cryptochromes from Cyclobutane Pyrimidine Dimer Photolyases by Affecting the Proteins' Preferences for Reduced FAD.

PubMed

Xu, Lei; Wen, Bin; Wang, Yuan; Tian, Changqing; Wu, Mingcai; Zhu, Guoping

2017-06-19

Cryptochromes (CRYs) and photolyases belong to the cryptochrome/photolyase family (CPF). Reduced FAD is essential for photolyases to photorepair UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts in DNA. In Drosophila CRY (dCRY, a type I animal CRY), FAD is converted to the anionic radical but not to the reduced state upon illumination, which might induce a conformational change in the protein to relay the light signal downstream. To explore the foundation of these differences, multiple sequence alignment of 650 CPF protein sequences was performed. We identified a site facing FAD (Ala377 in Escherichia coli CPD photolyase and Val415 in dCRY), hereafter referred to as "site 377", that was distinctly conserved across these sequences: CPD photolyases often had Ala, Ser, or Asn at this site, whereas animal CRYs had Ile, Leu, or Val. The binding affinity for reduced FAD, but not the photorepair activity of E. coli photolyase, was dramatically impaired when replacing Ala377 with any of the three CRY residues. Conversely, in V415S and V415N mutants of dCRY, FAD was photoreduced to its fully reduced state after prolonged illumination, and light-dependent conformational changes of these mutants were severely inhibited. We speculate that the residues at site 377 play a key role in the different preferences of CPF proteins for reduced FAD, which differentiate animal CRYs from CPD photolyases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Resonance Raman spectroscopic investigation of the light-harvesting chromophore in escherichia coli photolyase and Vibrio cholerae cryptochrome-1.

PubMed

Sokolova, Olga; Cecala, Christine; Gopal, Anand; Cortazar, Frank; McDowell-Buchanan, Carla; Sancar, Aziz; Gindt, Yvonne M; Schelvis, Johannes P M

2007-03-27

Photolyases and cryptochromes are flavoproteins that belong to the class of blue-light photoreceptors. They usually bind two chromophores: flavin adenine dinucleotide (FAD), which forms the active site, and a light-harvesting pigment, which is a 5,10-methenyltetrahydrofolate polyglutamate (MTHF) in most cases. In Escherichia coli photolyase (EcPhr), the MTHF cofactor is present in substoichiometric amounts after purification, while in Vibrio cholerae cryptochrome-1 (VcCry1) the MTHF cofactor is bound more strongly and is present at stoichiometric levels after purification. In this paper, we have used resonance Raman spectroscopy to monitor the effect of loss of MTHF on the protein-FAD interactions in EcPhr and to probe the protein-MTHF interactions in both EcPhr and VcCry1. We find that removal of MTHF does not perturb protein-FAD interactions, suggesting that it may not affect the physicochemical properties of FAD in EcPhr. Our data demonstrate that the pteridine ring of MTHF in EcPhr has different interactions with the protein matrix than that of MTHF in VcCry1. Comparison to solution resonance Raman spectra of MTHF suggests that the carbonyl of its pteridine ring in EcPhr experiences stronger hydrogen bonding and a more polar environment than in VcCry1, but that hydrogen bonding to the pteridine ring amine hydrogens is stronger in VcCry-1. These differences in hydrogen bonding may account for the higher binding affinity of MTHF in VcCry1 compared to EcPhr.

Tradeoff between reproduction and resistance evolution to Bt-toxin in Helicoverpa armigera: regulated by vitellogenin gene expression.

PubMed

Zhang, W N; Xiao, H J; Liang, G M; Guo, Y Y; Wu, K M

2014-08-01

Evolution of resistance to insecticides usually has fitness tradeoffs associated with adaptation to the stress. The basic regulation mechanism of tradeoff between reproduction and resistance evolution to Bacillus thuringiensis (Bt) toxin in the cotton bollworm, Helicoverpa armigera (Ha), based on the vitellogenin (Vg) gene expression was analyzed here. The full-length cDNA of the Vg gene HaVg (JX504706) was cloned and identified. HaVg has 5704 base pairs (bp) with an open reading frame (ORF) of 5265 bp, which encoded 1756 amino acid protein with a predicted molecular mass of 197.28 kDa and a proposed isoelectric point of 8.74. Sequence alignment analysis indicated that the amino acid sequence of HaVg contained all of the conserved domains detected in the Vgs of the other insects and had a high similarity with the Vgs of the Lepidoptera insects, especially Noctuidae. The resistance level to Cry1Ac Bt toxin and relative HaVg mRNA expression levels among the following four groups: Cry1Ac-susceptible strain (96S), Cry1Ac-resistant strain fed on artificial diet with Bt toxin for 135 generations (BtR stands for the Cry1Ac Bt resistance), progeny of the Cry1Ac-resistant strain with a non-Bt-toxin artificial diet for 38 generations (CK1) and the direct descendants of the 135th-generation resistant larvae which were fed on an artificial diet without the Cry1Ac protein (CK2) were analyzed. Compared with the 96S strain, the resistance ratios of the BtR strain, the CK1 strain and the CK2 strain were 2917.15-, 2.15- and 2037.67-fold, respectively. The maximum relative HaVg mRNA expression levels of the BtR strain were approximately 50% less than that of the 96S strain, and the coming of maximum expression was delayed for approximately 4 days. The overall trend of the HaVg mRNA expression levels in the CK1 strain was similar to that in the 96S strain, and the overall trend of the HaVg mRNA expression levels in the CK2 strain was similar to that in the BtR strain. Our results suggest that the changes in reproduction due to the Bt-toxin resistance evolution in the BtR strain may be regulated by the Vg gene expression. The down-regulation of HaVg at the early stages resulted in a period of delayed reproduction and decreased fecundity in the BtR strain. This performance disappeared when the Bt-toxin selection pressure was lost.

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

DOE PAGES

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany; ...

2017-01-31

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin

USDA-ARS?s Scientific Manuscript database

Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against some coleopteran pests, but improvements are needed in both efficacy and “time to kill” for Cry toxins to become valuable tools for use in integrated pest management. To gain insight into Bt intoxication of Coleoptera, we perfo...

Bacillus thuringiensis Cry1Ia10 and Vip3Aa protein interactions and their toxicity in Spodoptera spp. (Lepidoptera).

PubMed

Bergamasco, V B; Mendes, D R P; Fernandes, O A; Desidério, J A; Lemos, M V F

2013-02-01

The polyphagous pests belonging to the genus Spodoptera are considered to be among the most important causes of damage and are widely distributed throughout the Americas'. Due to the extensive use of genetically modified plants containing Bacillus thuringiensis genes that code for insecticidal proteins, resistant insects may arise. To prevent the development of resistance, pyramided plants, which express multiple insecticidal proteins that act through distinct mode of actions, can be used. This study analyzed the mechanisms of action for the proteins Cry1Ia10 and Vip3Aa on neonatal Spodoptera frugiperda, Spodoptera albula, Spodoptera eridania and Spodoptera cosmioides larvae. The interactions of these toxins with receptors on the intestinal epithelial membrane were also analyzed by binding biotinylated toxins to brush border membrane vesicles (BBMVs) from the intestines of these insects. A putative receptor of approximately 65 kDa was found by ligand blotting in all of these species. In vitro competition assays using biotinylated proteins have indicated that Vip3Aa and Cry1Ia10 do not compete for the same receptor for S. frugiperda, S. albula and S. cosmioides and that Vip3Aa was more efficient than Cry1Ia10 when tested individually, by bioassays. A synergistic effect of the toxins in S. frugiperda, S. albula and S. cosmioides was observed when they were combined. However, in S. eridania, Cry1Ia10 and Vip3Aa might compete for the same receptor and through bioassays Cry1Ia10 was more efficient than Vip3Aa and showed an antagonistic effect when the proteins were combined. These results suggest that using these genes to develop pyramided plants may not prove effective in preventing the development of resistance in S. eridiana. Copyright © 2012 Elsevier Inc. All rights reserved.

Influences of Cry1Ac broccoli on larval survival and oviposition of diamondback moth.

PubMed

Yi, Dengxia; Cui, Shusong; Yang, Limei; Fang, Zhiyuan; Liu, Yumei; Zhuang, Mu; Zhang, Yangyong

2015-01-01

Larval survival and oviposition behavior of three genotypes of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), (homozygous Cry1Ac-susceptibile, Cry1Ac-resistant, and their F1 hybrids), on transgenic Bacillus thuringiensis (Bt) broccoli expressing different levels of Cry1Ac protein were evaluated in laboratory. These Bt broccoli lines were designated as relative low, medium, and high, respectively, according to the Cry1Ac content. Untransformed brocccoli plants were used as control. Larval survival of diamondback moth on non-Bt leaves was not significantly different among the three genotypes. The Cry1Ac-resistant larvae could survive on the low level of Bt broccoli plants, while Cry1Ac-susceptible and F1 larvae could not survive on them. The three genotypes of P. xylostella larvae could not survive on medium and high levels of Bt broccoli. In oviposition choice tests, there was no significant difference in the number of eggs laid by the three P. xylostella genotypes among different Bt broccoli plants. The development of Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella on intact Bt plants was also tested in greenhouse. All susceptible P. xylostella larvae died on all Bt plants, while resistant larvae could survive on broccoli, which expresses low Cry1Ac protein under greenhouse conditions. The results of the greenhouse trials were similar to that of laboratory tests. This study indicated that high dose of Bt toxins in broccoli cultivars or germplasm lines is required for effective resistance management. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Dual mode of action of Bt proteins: protoxin efficacy against resistant insects

PubMed Central

Tabashnik, Bruce E.; Zhang, Min; Fabrick, Jeffrey A.; Wu, Yidong; Gao, Meijing; Huang, Fangneng; Wei, Jizhen; Zhang, Jie; Yelich, Alexander; Unnithan, Gopalan C.; Bravo, Alejandra; Soberón, Mario; Carrière, Yves; Li, Xianchun

2015-01-01

Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops. PMID:26455902

Cry-Bt identifier: a biological database for PCR detection of Cry genes present in transgenic plants.

PubMed

Singh, Vinay Kumar; Ambwani, Sonu; Marla, Soma; Kumar, Anil

2009-10-23

We describe the development of a user friendly tool that would assist in the retrieval of information relating to Cry genes in transgenic crops. The tool also helps in detection of transformed Cry genes from Bacillus thuringiensis present in transgenic plants by providing suitable designed primers for PCR identification of these genes. The tool designed based on relational database model enables easy retrieval of information from the database with simple user queries. The tool also enables users to access related information about Cry genes present in various databases by interacting with different sources (nucleotide sequences, protein sequence, sequence comparison tools, published literature, conserved domains, evolutionary and structural data). http://insilicogenomics.in/Cry-btIdentifier/welcome.html.

40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance. 174.510 Section 174.510 Protection of... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.510 Bacillus thuringiensis...

Minnesota field population of western corn rootworm (Coleoptera: Chrysomelidae) shows incomplete resistance to Cry34Ab1/Cry35Ab1 and Cry3Bb1

USDA-ARS?s Scientific Manuscript database

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is commonly managed with corn (Zea mays L.) expressing insecticidal proteins from the bacteria Bacillus thuringiensis (Bt). Under laboratory conditions, populations of western corn rootworm have been selected for resistance to each c...

Development and characterization of MIR604 resistance in a western corn rootworm population (Coleoptera: Chrysomelidae)

USDA-ARS?s Scientific Manuscript database

mCry3A is one of only four proteins licensed for commercial use in Diabrotica control. Utilizing a colony of western corn rootworm, Diabrotica virgifera virgifera LeConte, selected for resistance to mCry3A, we evaluated how mCry3A resistance was inherited and whether fitness costs were associated wi...

Down-regulation of aminopeptidase N and ABC transporter subfamily G transcripts in Cry1Ab and Cry1Ac resistant Asian corn borer, Ostrina furnacalis (Lepidoptera: Crambidae)

USDA-ARS?s Scientific Manuscript database

Bacillus thuringiensis (Bt) crystalline protein (Cry) toxins cause mortality by a mechanism involving pore formation or signal transduction following toxin binding to receptors along the midgut lumen of susceptible insects, but this mechanism and mutations therein that lead to resistance remain poor...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

A Genomic and Proteomic Approach to Identify and Quantify the Expressed Bacillus thuringiensis Proteins in the Supernatant and Parasporal Crystal.

PubMed

Gomis-Cebolla, Joaquín; Scaramal Ricietto, Ana Paula; Ferré, Juan

2018-05-10

The combined analysis of genomic and proteomic data allowed us to determine which cry and vip genes are present in a Bacillus thuringiensis ( Bt ) isolate and which ones are being expressed. Nine Bt isolates were selected from Spanish collections of Bt based on their vip1 and vip2 gene content. As a first step, nine isolates were analyzed by PCR to select those Bt isolates that contained genes with the lowest similarity to already described vip1 and vip2 genes (isolates E-SE10.2 and O-V84.2). Two selected isolates were subjected to a combined genomic and proteomic analysis. The results showed that the Bt isolate E-SE10.2 codifies for two new vegetative proteins, Vip2Ac-like_1 and Sip1Aa-like_1, that do not show expression differences at 24 h vs. 48 h and are expressed in a low amount. The Bt isolate O-V84.2 codifies for three new vegetative proteins, Vip4Aa-like_1, Vip4Aa-like_2, and Vip2Ac-like_2, that are marginally expressed. The Vip4Aa-like_1 protein was two-fold more abundant at 24 h vs. 48 h, while the Vip4Aa-like_2 was detected only at 24 h. For Vip2Ac-like_2, no differences in expression were found at 24 h vs. 48 h. Moreover, the parasporal crystal of the E-SE10.2 isolate contains a single type of crystal protein, Cry23Aa-like, while the parasporal crystal from O-V84.2 contains three kinds of crystal proteins: 7.0⁻9.8% weight of Cry45Aa-like proteins, 35⁻37% weight of Cry32-like proteins and 2.8⁻4.3% weight of Cry73-like protein.

Differential protection of Cry1Fa toxin against Spodoptera frugiperda larval gut proteases by cadherin orthologs correlates with increased synergism.

PubMed

Rahman, Khalidur; Abdullah, Mohd Amir F; Ambati, Suresh; Taylor, Milton D; Adang, Michael J

2012-01-01

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.

Comparison and validation of methods to quantify Cry1Ab toxin from Bacillus thuringiensis for standardization of insect bioassays.

PubMed

Crespo, André L B; Spencer, Terence A; Nekl, Emily; Pusztai-Carey, Marianne; Moar, William J; Siegfried, Blair D

2008-01-01

Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC(50) values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.

Effect of transgenic Bacillus thuringiensis rice lines on mortality and feeding behavior of rice stem borers (Lepidoptera: Crambidae).

PubMed

Chen, Hao; Zhang, Guoan; Zhang, Qifa; Lin, Yongjun

2008-02-01

Ten transgenic Bacillus thuringiensis Bt rice, Oryza sativa L., lines with different Bt genes (two Cry1Ac lines, three Cry2A lines, and five Cry9C lines) derived from the same variety Minghui 63 were evaluated in both the laboratory and the field. Bioassays were conducted by using the first instars of two main rice lepidopteran insect species: yellow stem borer, Scirpophaga incertulas (Walker) and Asiatic rice borer, Chilo suppressalis (Walker). All transgenic lines exhibited high toxicity to these two rice borers. Field evaluation results also showed that all transgenic lines were highly insect resistant with both natural infestation and manual infestation of the neonate larvae of S. incertulas compared with the nontransformed Minghui63. Bt protein concentrations in leaves of 10 transgenic rice lines were estimated by the sandwich enzyme-linked immunosorbent assay. The cry9C gene had the highest expression level, next was cry2A gene, and the cry1Ac gene expressed at the lowest level. The feeding behavior of 7-d-old Asiatic rice borer to three classes of Bt transgenic rice lines also was detected by using rice culm cuttings. The results showed that 7-d-old larvae of Asiatic rice borer have the capacity to distinguish Bt and non-Bt culm cuttings and preferentially fed on non-Bt cuttings. When only Bt culm cuttings with three classes of different Bt proteins (CrylAc, Cry2A, and Cry9C) were fed, significant distribution difference of 7-d-old Asiatic rice borer in culm cuttings of different Bt proteins also was found. In the current study, we evaluate different Bt genes in the same rice variety in both the laboratory and the field, and also tested feeding behavior of rice insect to these Bt rice. These data are valuable for the further development of two-toxin Bt rice and establishment of appropriate insect resistance management in the future.

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

PubMed

Agaisse, H; Lereclus, D

1994-08-01

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.

CHARACTERIZATION OF H-Y AND CR-Y ZEOLITE CATALYSTS DURING THE OXIDATIVE DESTRUCTION OF CFC11 AND CFC12

EPA Science Inventory

The long term stability-deactivation characteristics of two Y zeolite catalysts, namely H-Y and cation exchanged Cr-Y, were studied during the oxidative destruction of CFC11 and CFC12 feeds. Experiments were carried out at 300 degrees C and 500 h-1 space velocity. Properties of...

Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm.

PubMed

Wei, Jizhen; Liang, Gemei; Wang, Bingjie; Zhong, Feng; Chen, Lin; Khaing, Myint Myint; Zhang, Jie; Guo, Yuyuan; Wu, Kongming; Tabashnik, Bruce E

2016-01-01

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm

PubMed Central

Liang, Gemei; Wang, Bingjie; Zhong, Feng; Chen, Lin; Khaing, Myint Myint; Zhang, Jie; Guo, Yuyuan; Wu, Kongming; Tabashnik, Bruce E.

2016-01-01

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins. PMID:27257885

Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.).

PubMed

Zheng, Si-Jun; Henken, Betty; de Maagd, Ruud A; Purwito, Agus; Krens, Frans A; Kik, Chris

2005-06-01

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.

Characterization of Bacillus thuringiensis Berl. indigenous from soil and its potency as biological agents of Spodoptera litura (Lepidoptera: Noctuidae)

NASA Astrophysics Data System (ADS)

Pujiastuti, Y.; Astuti, D. T.; Afriyani, S. R.; Suparman, S.; Irsan, C.; Sembiring, E. R.; Nugraha, S.; Mulawarman; Damiri, N.

2018-01-01

The objective of the study was to characterize the molecular weight of protein in order to be utilized as biological agent of S. litura and their cell or spores production. To investigate the molecular weight of protein was done by SDS-Page electrophoresis. Growth media used for producing B. thuringiensis were T3, LB broth and agricultural waste. The results showed that the molecular weight of protein ranged from 37 to 140 kDa. In DLM and DLKK23 isolates were found ranging from 37 to 40 kDa and from 110 to 130 kDa, respectively. KJ3R5 and KJ3P1 isolates were obtained having three protein bands ranging from 43 to 45, 73 to 80 and 110 to 130 kDa and 45-50, 75-80 and 130-140 kDa, respectively. It was predicted that isolates B. thuringiensis were belonging to Cry IA, Cry IIA, Cry IVC and Cry15c. These crystal proteins were toxic to S. litura. There was no protein bands found in the two last isolates (KJ3R3 and KJ3J4). Production of spore after sporulation in agricultural waste media ranged from 0.5 to 106 - 2.67 x 107 spores/ml showing medium level of toxicity to S. litura.

Construction of a biodynamic model for Cry protein production studies.

PubMed

Navarro-Mtz, Ana Karin; Pérez-Guevara, Fermín

2014-12-01

Mathematical models have been used from growth kinetic simulation to gen regulatory networks prediction for B. thuringiensis culture. However, this culture is a time dependent dynamic process where cells physiology suffers several changes depending on the changes in the cell environment. Therefore, through its culture, B. thuringiensis presents three phases related with the predominance of three major metabolic pathways: vegetative growth (Embded-Meyerhof-Parnas pathway), transition (γ-aminobutiric cycle) and sporulation (tricarboxylic acid cycle). There is not available a mathematical model that relates the different stages of cultivation with the metabolic pathway active on each one of them. Therefore, in the present study, and based on published data, a biodynamic model was generated to describe the dynamic of the three different phases based on their major metabolic pathways. The biodynamic model is used to study the interrelation between the different culture phases and their relationship with the Cry protein production. The model consists of three interconnected modules where each module represents one culture phase and its principal metabolic pathway. For model validation four new fermentations were done showing that the model constructed describes reasonably well the dynamic of the three phases. The main results of this model imply that poly-β-hydroxybutyrate is crucial for endospore and Cry protein production. According to the yields of dipicolinic acid and Cry from poly-β-hydroxybutyrate, calculated with the model, the endospore and Cry protein production are not just simultaneous and parallel processes they are also competitive processes.

Regional brain responses in nulliparous women to emotional infant stimuli.

PubMed

Montoya, Jessica L; Landi, Nicole; Kober, Hedy; Worhunsky, Patrick D; Rutherford, Helena J V; Mencl, W Einar; Mayes, Linda C; Potenza, Marc N

2012-01-01

Infant cries and facial expressions influence social interactions and elicit caretaking behaviors from adults. Recent neuroimaging studies suggest that neural responses to infant stimuli involve brain regions that process rewards. However, these studies have yet to investigate individual differences in tendencies to engage or withdraw from motivationally relevant stimuli. To investigate this, we used event-related fMRI to scan 17 nulliparous women. Participants were presented with novel infant cries of two distress levels (low and high) and unknown infant faces of varying affect (happy, sad, and neutral) in a randomized, counter-balanced order. Brain activation was subsequently correlated with scores on the Behavioral Inhibition System/Behavioral Activation System scale. Infant cries activated bilateral superior and middle temporal gyri (STG and MTG) and precentral and postcentral gyri. Activation was greater in bilateral temporal cortices for low- relative to high-distress cries. Happy relative to neutral faces activated the ventral striatum, caudate, ventromedial prefrontal, and orbitofrontal cortices. Sad versus neutral faces activated the precuneus, cuneus, and posterior cingulate cortex, and behavioral activation drive correlated with occipital cortical activations in this contrast. Behavioral inhibition correlated with activation in the right STG for high- and low-distress cries relative to pink noise. Behavioral drive correlated inversely with putamen, caudate, and thalamic activations for the comparison of high-distress cries to pink noise. Reward-responsiveness correlated with activation in the left precentral gyrus during the perception of low-distress cries relative to pink noise. Our findings indicate that infant cry stimuli elicit activations in areas implicated in auditory processing and social cognition. Happy infant faces may be encoded as rewarding, whereas sad faces activate regions associated with empathic processing. Differences in motivational tendencies may modulate neural responses to infant cues.

A cry in the dark: depressed mothers show reduced neural activation to their own infant’s cry

PubMed Central

Ablow, Jennifer C.

2012-01-01

This study investigated depression-related differences in primiparous mothers’ neural response to their own infant’s distress cues. Mothers diagnosed with major depressive disorder (n = 11) and comparison mothers with no diagnosable psychopathology (n = 11) were exposed to their own 18-months-old infant’s cry sound, as well as unfamiliar infant’s cry and control sound, during functional neuroimaging. Depressed mothers’ response to own infant cry greater than other sounds was compared to non-depressed mothers’ response in the whole brain [false discovery rate (FDR) corrected]. A continuous measure of self-reported depressive symptoms (CESD) was also tested as a predictor of maternal response. Non-depressed mothers activated to their own infant’s cry greater than control sound in a distributed network of para/limbic and prefrontal regions, whereas depressed mothers as a group failed to show activation. Non-depressed compared to depressed mothers showed significantly greater striatal (caudate, nucleus accumbens) and medial thalamic activation. Additionally, mothers with lower depressive symptoms activated more strongly in left orbitofrontal, dorsal anterior cingulate and medial superior frontal regions. Non-depressed compared to depressed mothers activated uniquely to own infant greater than other infant cry in occipital fusiform areas. Disturbance of these neural networks involved in emotional response and regulation may help to explain parenting deficits in depressed mothers. PMID:21208990

F2 screen, inheritance and cross-resistance of field-derived Vip3A resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) collected from Louisiana, USA.

PubMed

Yang, Fei; Morsello, Shannon; Head, Graham P; Sansone, Chris; Huang, Fangneng; Gilreath, Ryan T; Kerns, David L

2017-11-28

Fall armyworm, Spodoptera frugiperda, is a target pest of the Vip3A protein used in pyramided Bt corn and cotton in the USA. In this study, we provide the first documentation of a resistance allele conferring Vip3A resistance in a field-derived population of S. frugiperda from the USA, and characterize its inheritance and cross-resistance. An F 2 screen with 104 two-parent families generated from a field collection of S. frugiperda in Louisiana, USA, resulted in one family carrying a Vip3A resistance allele. The Vip3A-resistant strain (RR) derived from the two-parent family showed a high level of resistance to Vip3A in both diet and whole-plant bioassays, with a resistance ratio of >632.0-fold relative to a susceptible population (SS) based on diet-overlay bioassays. The inheritance of Vip3A resistance was monogenic, autosomal and recessive. Furthermore, the Vip3A resistance conferred no cross-resistance to Cry1F, Cry2Ab2 or Cry2Ae purified proteins, with resistance ratios of 3.5, 5.0 and 1.1, respectively. These findings provide valuable information for characterizing Vip3A resistance, resistance monitoring, and developing effective resistance management strategies for the sustainable use of the Vip3A technology. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

PubMed Central

Rahman, Khalidur; Abdullah, Mohd Amir F.; Ambati, Suresh; Taylor, Milton D.

2012-01-01

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin. PMID:22081566

75 FR 52329 - Corn Event MON 863 and MON 863 x MON 810; Product Cancellation Order for Certain Pesticide...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-25

... MON 863 and MON 863 x MON 810 seed through July 1, 2011, for planting by July 1, 2011. 3. Existing... certain products containing the pesticides, Bacillus thuringiensis Cry3Bb1 protein and the genetic... thuringiensis Cry1Ab protein and the genetic material necessary for its production (vector PV-ZMCT01) in MON 810...

Inheritance of Cry1F resistance, cross-resistance and frequency of resistant alleles in Spodoptera frugiperda (Lepidoptera: Noctuidae).

PubMed

Vélez, A M; Spencer, T A; Alves, A P; Moellenbeck, D; Meagher, R L; Chirakkal, H; Siegfried, B D

2013-12-01

Transgenic maize, Zea maize L., expressing the Cry1F protein from Bacillus thuringiensis has been registered for Spodoptera frugiperda (J. E. Smith) control since 2003. Unexpected damage to Cry1F maize was reported in 2006 in Puerto Rico and Cry1F resistance in S. frugiperda was documented. The inheritance of Cry1F resistance was characterized in a S. frugiperda resistant strain originating from Puerto Rico, which displayed >289-fold resistance to purified Cry1F. Concentration-response bioassays of reciprocal crosses of resistant and susceptible parental populations indicated that resistance is recessive and autosomal. Bioassays of the backcross of the F1 generation crossed with the resistant parental strain suggest that a single locus is responsible for resistance. In addition, cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry2Aa and Vip3Aa was assessed in the Cry1F-resistant strain. There was no significant cross-resistance to Cry1Aa, Cry1Ba and Cry2Aa, although only limited effects were observed in the susceptible strain. Vip3Aa was highly effective against susceptible and resistant insects indicating no cross-resistance with Cry1F. In contrast, low levels of cross-resistance were observed for both Cry1Ab and Cry1Ac. Because the resistance is recessive and conferred by a single locus, an F1 screening assay was used to measure the frequency of Cry1F-resistant alleles from populations of Florida and Texas in 2010 and 2011. A total frequency of resistant alleles of 0.13 and 0.02 was found for Florida and Texas populations, respectively, indicating resistant alleles could be found in US populations, although there have been no reports of reduced efficacy of Cry1F-expressing plants.

Identification of Small Molecule Activators of Cryptochrome

PubMed Central

Hirota, Tsuyoshi; Lee, Jae Wook; St. John, Peter C.; Sawa, Mariko; Iwaisako, Keiko; Noguchi, Takako; Pongsawakul, Pagkapol Y.; Sonntag, Tim; Welsh, David K.; Brenner, David A.; Doyle, Francis J.; Schultz, Peter G.; Kay, Steve A.

2013-01-01

Impairment of the circadian clock has been associated with numerous disorders, including metabolic disease. Although small molecules that modulate clock function might offer therapeutic approaches to such diseases, only a few compound have been identified that selectively target core clock proteins. From an unbiased cell-based circadian screen, we identified KL001, a small molecule that specifically interacts with cryptochrome (CRY). KL001 prevented ubiquitin-dependent degradation of CRY, resulting in lengthening of the circadian period. In combination with mathematical modeling, KL001 revealed that CRY1 and CRY2 share a similar functional role in the period regulation. Furthermore, KL001- mediated CRY stabilization inhibited glucagon-induced gluconeogenesis in primary hepatocytes. KL001 thus provides a tool to study the regulation of CRY-dependent physiology and aid development of clock-based therapeutics of diabetes. PMID:22798407

Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer.

PubMed

Cheng, X; Sardana, R; Kaplan, H; Altosaar, I

1998-03-17

Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and beta-glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific delta-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R0 and R1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0 plants. Bioassays with R1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.

Herbicide and insect resistant Bt cotton pollen assessment finds no detrimental effects on adult honey bees.

PubMed

Niu, Lin; Ma, Weihua; Lei, Chaoliang; Jurat-Fuentes, Juan Luis; Chen, Lizhen

2017-11-01

One important concern regarding the use of transgenic cotton expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) is its potential detrimental effect on non-target organisms. The honey bee (Apis mellifera) is the most important pollinator species worldwide and it is directly exposed to transgenic crops by the consumption of genetically modified (GM) pollen. However, the potential effects of Bt cotton on A. mellifera remain unclear. In the present study, we assessed the effects of two Bt cotton varieties; ZMSJ expressing the Cry1Ac and Cry2Ab insecticidal proteins, and ZMKCKC producing Cry1Ac and EPSPS, on A. mellifera. Feeding on pollen from two Bt cotton varieties led to detection of low levels of Cry toxins (<10 ng/g fresh weight) in the midgut of A. mellifera adults, yet expression of detoxification genes did not change significantly compared to feeding on non-Bt cotton. Binding assays showed no Cry1Ac or Cry2Ab binding to midgut brush border membrane proteins from A. mellifera adults. Taken together, these results support minimal risk for potential negative effects on A. mellifera by exposure to Bt cotton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transgenic rice plants expressing a fused protein of Cry1Ab/Vip3H has resistance to rice stem borers under laboratory and field conditions.

PubMed

Chen, Yang; Tian, Jun-Ce; Shen, Zhi-Chen; Peng, Yu-Fa; Hu, Cui; Guo, Yu-Yuan; Ye, Gong-Yin

2010-08-01

Six transgenic rice, Oryza sativa L., lines (G6H1, G6H2, G6H3, G6H4, G6H5, and G6H6) expressing a fused Cry1Ab/Vip3H protein, were evaluated for resistance against the Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), and the stem borer Sesamia inferens (Walker) (Lepidoptera: Noctuidae) in the laboratory and field. The bioassay results indicated that the mortality of Asiatic rice borer and S. inferens neonate larvae on six transgenic lines from seedling to filling stage was up to 100% at 168 h after infestation. The cumulative feeding area by Asiatic rice borer neonate larvae on all transgenic lines was significantly reduced compared with the untransformed parental 'Xiushui 110' rice. A 2-yr field evaluation showed that damage during the vegetative stage (deadheart) or during the reproductive stage (whitehead) caused by Asiatic rice borer and S. inferens for transgenic lines was much lower than the control. For three lines (G6H1, G6H2, and G6H6), no damage was found during the entire growing period. Estimation of fused Cry1Ab/Vip3H protein concentrations using PathoScreen kit for Bt-Cry1Ab/1Ac protein indicated that the expression levels of Cry1Ab protein both in main stems (within the average range of 0.006-0.073% of total soluble protein) and their flag leaves (within the average range of 0.001-0.038% of total soluble protein) were significantly different among six transgenic lines at different developmental stages. Both laboratory and field researches suggested that the transgenic rice lines have considerable potential for protecting rice from attack by both stem borers.

Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica).

PubMed

Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Rui-Fen; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

2013-04-01

MdCRY2 was isolated from apple fruit skin, and its function was analyzed in MdCRY2 transgenic Arabidopsis. The interaction between MdCRY2 and AtCOP1 was found by yeast two-hybrid and BiFC assays. Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development. Investigations of the structure and functions of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), pea (Pisum sativum), and rice (Oryza sativa). However, no data on the function of CRY2 are available in woody plants. In this study, we isolated a cryptochrome gene, MdCRY2, from apple (Malus domestica). The deduced amino acid sequences of MdCRY2 contain the conserved N-terminal photolyase-related domain and the flavin adenine dinucleotide (FAD) binding domain, as well as the C-terminal DQXVP-acidic-STAES (DAS) domain. Relationship analysis indicates that MdCRY2 shows the highest similarity to the strawberry FvCRY protein. The expression of MdCRY2 is induced by blue/UV-A light, which represents a 48-h circadian rhythm. To investigate the function of MdCRY2, we overexpressed the MdCRY2 gene in a cry2 mutant and wild type (WT) Arabidopsis, assessed the phenotypes of the resulting transgenic plants, and found that MdCRY2 functions to regulate hypocotyl elongation, root growth, flower initiation, and anthocyanin accumulation. Furthermore, we examined the interaction between MdCRY2 and AtCOP1 using a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. These data provide functional evidence for a role of blue/UV-A light-induced MdCRY2 in controlling photomorphogenesis in apple.

Expression of a germline human kappa chain-associated cross-reactive idiotype after in vitro and in vivo infection with Epstein-Barr virus.

PubMed

Silverman, G J; Carson, D A; Patrick, K; Vaughan, J H; Fong, S

1987-06-01

The mouse monoclonal antibody 17.109 recognizes a cross-reactive idiotype (CRI) associated with kappa IIIb light chains of human IgM-rheumatoid factor (RF) paraproteins. The 17.109 idiotypic determinant is encoded by one or a group of closely related V kappa genes. The association of the idiotype with IgM- and IgA-rheumatoid factors in certain autoimmune diseases necessitates an understanding of how human B lymphocytes can be induced to express the idiotype. To investigate the cellular expression of the 17.109 CRI, peripheral blood lymphocytes from normal donors were stimulated in vitro with Epstein-Barr virus (EBV) and pokeweed mitogen (PWM). EBV induced greater expression of IgM-associated 17.109 CRI than did PWM. The 17.109 CRI was preferentially associated with IgM rather than with IgG. In vivo EBV infection was studied in college students with infectious mononucleosis and displayed similar elevation of IgM-associated 17.109 CRI in sera obtained at presentation of clinical illness. Later, IgM levels declined while IgG-associated 17.109 CRI rose. The 17.109 idiotype was unrelated to antibodies against the Epstein-Barr virus nuclear antigen and the viral capsid antigen and was probably due to generalized activation of early B cells. These observations support the hypothesis that the 17.109 CRI is expressed by in vitro and in vivo EBV-infected cells. The 17.109 idiotype identifies a highly conserved V kappa gene product, which is expressed preferentially after EBV infection, but not exclusively with RF autoantibodies.

Fitness Costs Related To Selection for Resistance to the Cry3Bb1 Protein in a Genetically Diverse Population of Non-diapausing Western Corn Rootworm

USDA-ARS?s Scientific Manuscript database

The western corn rootworm (Diabrotica virgifera virgifera LeConte) is an important pest of maize in North America. Since approved for commercial use in 2003, the acreage planted to maize (Zea mays L.) expressing the Bt derived Cry3Bb1 protein has significantly increased each year in the United State...

Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence

Treesearch

Hyun-woo Park; Baoxue Ge; Leah S. Bauer; Brian A. Federici

1998-01-01

The insecticidal activity of Bacillus thuringiensis strains toxic to coleopterous insects is due to Cry3 proteins assembled into small rectangular crystals. Toxin synthesis in these strains is dependent primarily upon a promoter that is active in the stationary phase and a STAB-SD sequence that stabilizes the cry3 transcript-ribosome complex. Here we show that...

Binding Site Concentration Explains the Differential Susceptibility of Chilo suppressalis and Sesamia inferens to Cry1A-Producing Rice

PubMed Central

Han, Chao; Liu, Zewen; Chen, Fajun; Hou, Maolin; Peng, Yufa

2014-01-01

Chilo suppressalis and Sesamia inferens are two important lepidopteran rice pests that occur concurrently during outbreaks in paddy fields in the main rice-growing areas of China. Previous and current field tests demonstrate that the transgenic rice line Huahui 1 (HH1) producing a Cry1Ab-Cry1Ac hybrid toxin from the bacterium Bacillus thuringiensis reduces egg and larval densities of C. suppressalis but not of S. inferens. This differential susceptibility to HH1 rice correlates with the reduced susceptibility to Cry1Ab and Cry1Ac toxins in S. inferens larvae compared to C. suppressalis larvae. The goal of this study was to identify the mechanism responsible for this differential susceptibility. In saturation binding assays, both Cry1Ab and Cry1Ac toxins bound with high affinity and in a saturable manner to midgut brush border membrane vesicles (BBMV) from C. suppressalis and S. inferens larvae. While binding affinities were similar, a dramatically lower concentration of Cry1A toxin binding sites was detected for S. inferens BBMV than for C. suppressalis BBMV. In contrast, no significant differences between species were detected for Cry1Ca toxin binding to BBMV. Ligand blotting detected BBMV proteins binding Cry1Ac or Cry1Ca toxins, some of them unique to C. suppressalis or S. inferens. These data support that reduced Cry1A binding site concentration is associated with a lower susceptibility to Cry1A toxins and HH1 rice in S. inferens larvae than in C. suppressalis larvae. Moreover, our data support Cry1Ca as a candidate for pyramiding efforts with Cry1A-producing rice to extend the activity range and durability of this technology against rice stem borers. PMID:24928872

Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals

PubMed Central

Nießner, Christine; Denzau, Susanne; Malkemper, Erich Pascal; Gross, Julia Christina; Burda, Hynek; Winklhofer, Michael; Peichl, Leo

2016-01-01

Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown, and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae, and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein. PMID:26898837

Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals.

PubMed

Nießner, Christine; Denzau, Susanne; Malkemper, Erich Pascal; Gross, Julia Christina; Burda, Hynek; Winklhofer, Michael; Peichl, Leo

2016-02-22

Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown, and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae, and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein.

Targeted Quantitative Screening of Chromosome 18 Encoded Proteome in Plasma Samples of Astronaut Candidates.

PubMed

Kopylov, Artur T; Ilgisonis, Ekaterina V; Moysa, Alexander A; Tikhonova, Olga V; Zavialova, Maria G; Novikova, Svetlana E; Lisitsa, Andrey V; Ponomarenko, Elena A; Moshkovskii, Sergei A; Markin, Andrey A; Grigoriev, Anatoly I; Zgoda, Victor G; Archakov, Alexander I

2016-11-04

This work was aimed at estimating the concentrations of proteins encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy male volunteers (aged 20-47). These young persons have been certified by the medical evaluation board as healthy subjects ready for space flight training. Over 260 stable isotope-labeled peptide standards (SIS) were synthesized to perform the measurements of proteins encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed an estimate of the levels of 84 of 276 proteins encoded by Chr 18. These proteins were quantified in whole and depleted plasma samples. Concentration of the proteins detected varied from 10 -6 M (transthyretin, P02766) to 10 -11 M (P4-ATPase, O43861). A minor part of the proteins (mostly representing intracellular proteins) was characterized by extremely high inter individual variations. The results provide a background for studies of a potential biomarker in plasma among proteins encoded by Chr 18. The SRM raw data are available in ProteomeXchange repository (PXD004374).

Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure.

PubMed

Hu, Yan; Nguyen, Thanh-Thanh; Lee, Alice C Y; Urban, Joseph F; Miller, Melanie M; Zhan, Bin; Koch, David J; Noon, Jason B; Abraham, Ambily; Fujiwara, Ricardo Toshio; Bowman, Dwight D; Ostroff, Gary R; Aroian, Raffi V

2018-05-04

Hookworms are intestinal nematode parasites that infect nearly half a billion people and are globally one of the most important contributors to iron-deficiency anemia. These parasites have significant impacts in developing children, pregnant women and working adults. Of all the soil-transmitted helminths or nematodes (STNs), hookworms are by far the most important, with disease burdens conservatively estimated at four million DALYs (Disability-Adjusted Life Years) and with productivity losses of up to US$139 billion annually. To date, mainly one drug, albendazole is used for hookworm therapy in mass drug administration, which has on average ∼80% cure rate that is lower (<40%) in some places. Given the massive numbers of people needing treatment, the threat of parasite resistance, and the inadequacy of current treatments, new and better cures against hookworms are urgently needed. Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis (Bt) has demonstrated good efficacy against Ancylostoma ceylanicum hookworm infections in hamsters. Here we broaden studies of Cry5B to include tests against infections of Ancylostoma caninum hookworms in dogs and against infections of the dominant human hookworm, Necator americanus, in hamsters. We show that Cry5B is highly effective against all hookworm parasites tested in all models. Neutralization of stomach acid improves Cry5B efficacy, which will aid in practical application of Cry5B significantly. Importantly, we also demonstrate that the anti-nematode therapeutic efficacy of Cry5B is independent of the host immune system and is not itself negated by repeated dosing. This study indicates that Bt Cry5B is a pan-hookworm anthelmintic with excellent properties for use in humans and other animals. Copyright © 2018. Published by Elsevier Ltd.

Field Performance of Bt Eggplants (Solanum melongena L.) in the Philippines: Cry1Ac Expression and Control of the Eggplant Fruit and Shoot Borer (Leucinodes orbonalis Guenée)

PubMed Central

Hautea, Desiree M.; Taylo, Lourdes D.; Masanga, Anna Pauleen L.; Sison, Maria Luz J.; Narciso, Josefina O.; Quilloy, Reynaldo B.; Hautea, Randy A.; Shotkoski, Frank A.; Shelton, Anthony M.

2016-01-01

Plants expressing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling insect pests in maize and cotton globally. However, there are few Bt vegetable crops. Eggplant (Solanum melongena) is a popular vegetable grown throughout Asia that is heavily treated with insecticides to control the eggplant fruit and shoot borer, Leucinodes orbonalis (EFSB). Herein we provide the first publicly available data on field performance in Asia of eggplant engineered to produce the Cry1Ac protein. Replicated field trials with five Bt eggplant open-pollinated (OP) lines from transformation event EE-1 and their non-Bt comparators were conducted over three cropping seasons in the Philippines from 2010–2012. Field trials documented levels of Cry1Ac protein expressed in plants and evaluated their efficacy against the primary target pest, EFSB. Cry1Ac concentrations ranged from 0.75–24.7 ppm dry weight with the highest in the terminal leaves (or shoots) and the lowest in the roots. Cry1Ac levels significantly increased from the vegetative to the reproductive stage. Bt eggplant lines demonstrated excellent control of EFSB. Pairwise analysis of means detected highly significant differences between Bt eggplant lines and their non-Bt comparators for all field efficacy parameters tested. Bt eggplant lines demonstrated high levels of control of EFSB shoot damage (98.6–100%) and fruit damage (98.1–99.7%) and reduced EFSB larval infestation (95.8–99.3%) under the most severe pest pressure during trial 2. Moths that emerged from larvae collected from Bt plants in the field and reared in their Bt eggplant hosts did not produce viable eggs or offspring. These results demonstrate that Bt eggplant lines containing Cry1Ac event EE-1 provide outstanding control of EFSB and can dramatically reduce the need for conventional insecticides. PMID:27322533

A Spodoptera exigua Cadherin Serves as a Putative Receptor for Bacillus thuringiensis Cry1Ca Toxin and Shows Differential Enhancement of Cry1Ca and Cry1Ac Toxicity

PubMed Central

Ren, Xiang-Liang; Chen, Rui-Rui; Zhang, Ying; Ma, Yan; Cui, Jin-Jie; Han, Zhao-Jun; Mu, Li-Li

2013-01-01

Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca. PMID:23835184

Three cases of Creutzfeldt-Jakob disease with prion protein gene codon180 mutation presenting with pathological laughing and crying.

PubMed

Iwasaki, Yasushi

2012-08-15

Although there are no reports of pathological laughing and crying being observed in patients with Creutzfeldt-Jakob disease (CJD), the author experienced three patients with CJD with prion protein gene codon180 mutation (V180I CJD) who showed this characteristic clinical finding. This finding was observed from the early disease stage in all 3 patients and continued for several months. Startle reaction was also remarkable in all patients, although myoclonus was generally mild. The dissociation between the startle reaction and myoclonus was suspected to be another feature of V180I CJD. The pathological laughing and crying co-occured with the startle reaction and stopped right before the onset of akinetic mutism, and the degree of both symptoms was almost parallel during this period. On the basis of MRI and autopsy findings, pathological laughing and crying was suspected of being induced by the widespread cerebral cortical involvement that is characteristic of V180I CJD. From the present observations, the author speculated that pathological laughing and crying may be a comparatively frequent observation in V180I CJD patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

PubMed Central

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-01-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression. PMID:22434878

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

PubMed

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-07-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

Bacillus thuringiensis-derived Cry5B Has Potent Anthelmintic Activity against Ascaris suum

PubMed Central

Miller, Melanie M.; Scheib, Ulrike; Yiu, Ying Y.; Aroian, Raffi V.

2013-01-01

Ascaris suum and Ascaris lumbricoides are two closely related geo-helminth parasites that ubiquitously infect pigs and humans, respectively. Ascaris suum infection in pigs is considered a good model for A. lumbricoides infection in humans because of a similar biology and tissue migration to the intestines. Ascaris lumbricoides infections in children are associated with malnutrition, growth and cognitive stunting, immune defects, and, in extreme cases, life-threatening blockage of the digestive tract and aberrant migration into the bile duct and peritoneum. Similar effects can be seen with A. suum infections in pigs related to poor feed efficiency and performance. New strategies to control Ascaris infections are needed largely due to reduced treatment efficacies of current anthelmintics in the field, the threat of resistance development, and the general lack of new drug development for intestinal soil-transmitted helminths for humans and animals. Here we demonstrate for the first time that A. suum expresses the receptors for Bacillus thuringiensis crystal protein and novel anthelmintic Cry5B, which has been previously shown to intoxicate hookworms and which belongs to a class of proteins considered non-toxic to vertebrates. Cry5B is able to intoxicate A. suum larvae and adults and triggers the activation of the p38 mitogen-activated protein kinase pathway similar to that observed with other nematodes. Most importantly, two moderate doses of 20 mg/kg body weight (143 nM/kg) of Cry5B resulted in a near complete cure of intestinal A. suum infections in pigs. Taken together, these results demonstrate the excellent potential of Cry5B to treat Ascaris infections in pigs and in humans and for Cry5B to work effectively in the human gastrointestinal tract. PMID:23818995

Influence of transgenic rice expressing a fused Cry1Ab/1Ac protein on frogs in paddy fields.

PubMed

Wang, Jia-Mei; Chen, Xiu-Ping; Liang, Yu-Yong; Zhu, Hao-Jun; Ding, Jia-Tong; Peng, Yu-Fa

2014-11-01

As genetic engineering in plants is increasingly used to control agricultural pests, it is important to determine whether such transgenic plants adversely affect non-target organisms within and around cultivated fields. The cry1Ab/1Ac fusion gene from Bacillus thuringiensis (Bt) has insecticidal activity and has been introduced into rice line Minghui 63 (MH63). We evaluated the effect of transgenic cry1Ab/1Ac rice (Huahui 1, HH1) on paddy frogs by comparing HH1 and MH63 rice paddies with and without pesticide treatment. The density of tadpoles in rice fields was surveyed at regular intervals, and Cry1Ab/1Ac protein levels were determined in tissues of tadpoles and froglets collected from the paddy fields. In addition, Rana nigromaculata froglets were raised in purse nets placed within these experimental plots. The survival, body weight, feeding habits, and histological characteristics of the digestive tract of these froglets were analyzed. We found that the tadpole density was significantly decreased immediately after pesticide application, and the weight of R. nigromaculata froglets of pesticide groups was significantly reduced compared with no pesticide treatment, but we found no differences between Bt and non-Bt rice groups. Moreover, no Cry1Ab/1Ac protein was detected in tissue samples collected from 192 tadpoles and froglets representing all four experimental groups. In addition, R. nigromaculata froglets raised in purse seines fed primarily on stem borer and non-target insects, and showed no obvious abnormality in the microstructure of their digestive tracts. Based on these results, we conclude that cultivation of transgenic cry1Ab/1Ac rice does not adversely affect paddy frogs.

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

PubMed

Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

2006-09-01

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

Effective dominance of resistance of Spodoptera frugiperda to Bt maize and cotton varieties: implications for resistance management

PubMed Central

Horikoshi, Renato J.; Bernardi, Daniel; Bernardi, Oderlei; Malaquias, José B.; Okuma, Daniela M.; Miraldo, Leonardo L.; Amaral, Fernando S. de A. e; Omoto, Celso

2016-01-01

The resistance of fall armyworm (FAW), Spodoptera frugiperda, has been characterized to some Cry and Vip3A proteins of Bacillus thuringiensis (Bt) expressed in transgenic maize in Brazil. Here we evaluated the effective dominance of resistance based on the survival of neonates from selected Bt-resistant, heterozygous, and susceptible (Sus) strains of FAW on different Bt maize and cotton varieties. High survival of strains resistant to the Cry1F (HX-R), Cry1A.105/Cry2Ab (VT-R) and Cry1A.105/Cry2Ab/Cry1F (PW-R) proteins was detected on Herculex, YieldGard VT PRO and PowerCore maize. Our Vip3A-resistant strain (Vip-R) exhibited high survival on Herculex, Agrisure Viptera and Agrisure Viptera 3 maize. However, the heterozygous from HX-R × Sus, VT-R × Sus, PW-R × Sus and Vip-R × Sus had complete mortality on YieldGard VT PRO, PowerCore, Agrisure Viptera, and Agrisure Viptera 3, whereas the HX-R × Sus and Vip-R × Sus strains survived on Herculex maize. On Bt cotton, the HX-R, VT-R and PW-R strains exhibited high survival on Bollgard II. All resistant strains survived on WideStrike, but only PW-R and Vip-R × Sus survived on TwinLink. Our study provides useful data to aid in the understanding of the effectiveness of the refuge strategy for Insect Resistance Management of Bt plants. PMID:27721425

Effective dominance of resistance of Spodoptera frugiperda to Bt maize and cotton varieties: implications for resistance management

NASA Astrophysics Data System (ADS)

Horikoshi, Renato J.; Bernardi, Daniel; Bernardi, Oderlei; Malaquias, José B.; Okuma, Daniela M.; Miraldo, Leonardo L.; Amaral, Fernando S. De A. E.; Omoto, Celso

2016-10-01

The resistance of fall armyworm (FAW), Spodoptera frugiperda, has been characterized to some Cry and Vip3A proteins of Bacillus thuringiensis (Bt) expressed in transgenic maize in Brazil. Here we evaluated the effective dominance of resistance based on the survival of neonates from selected Bt-resistant, heterozygous, and susceptible (Sus) strains of FAW on different Bt maize and cotton varieties. High survival of strains resistant to the Cry1F (HX-R), Cry1A.105/Cry2Ab (VT-R) and Cry1A.105/Cry2Ab/Cry1F (PW-R) proteins was detected on Herculex, YieldGard VT PRO and PowerCore maize. Our Vip3A-resistant strain (Vip-R) exhibited high survival on Herculex, Agrisure Viptera and Agrisure Viptera 3 maize. However, the heterozygous from HX-R × Sus, VT-R × Sus, PW-R × Sus and Vip-R × Sus had complete mortality on YieldGard VT PRO, PowerCore, Agrisure Viptera, and Agrisure Viptera 3, whereas the HX-R × Sus and Vip-R × Sus strains survived on Herculex maize. On Bt cotton, the HX-R, VT-R and PW-R strains exhibited high survival on Bollgard II. All resistant strains survived on WideStrike, but only PW-R and Vip-R × Sus survived on TwinLink. Our study provides useful data to aid in the understanding of the effectiveness of the refuge strategy for Insect Resistance Management of Bt plants.

Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12.

PubMed

Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Amaro, Itzel; Ortíz, Ernesto; Becerril, Baltazar; Ibarra, Jorge E; Bravo, Alejandra; Soberón, Mario

2015-04-01

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of Bt Corn with Pyramided Genes on Efficacy and Insect Resistance Management for the Asian Corn Borer in China.

PubMed

Jiang, Fan; Zhang, Tiantao; Bai, Shuxiong; Wang, Zhenying; He, Kanglai

2016-01-01

A Bt corn hybrid (AcIe) with two Bt genes (cry1Ie and cry1Ac) was derived by breeding stack from line expressing Cry1Ie and a line expressing Cry1Ac. Efficacy of this pyramided Bt corn hybrid against the Asian corn borer (ACB), Ostrinia furnacalis, was evaluated. We conducted laboratory bioassays using susceptible and resistant ACB strains fed on artificial diet or fresh plant tissues. We also conducted field trials with artificial infestations of ACB neonates at the V6 and silk stages. The toxin-diet bioassay data indicated that mixtures of Cry1Ac and Cry1Ie proteins had synergistic insecticidal efficacy. The plant tissue bioassay data indicated that Bt corn hybrids expressing either a single toxin (Cry1Ac or Cry1Ie) or two toxins had high efficacy against susceptible ACB. Damage ratings in the field trials indicated that the Bt corn hybrids could effectively protect against 1st and the 2nd generation ACB in China. The hybrid line with two Bt genes showed a higher efficacy against ACB larvae resistant to Cry1Ac or CryIe than the hybrid containing one Bt gene, and the two gene hybrid would have increased potential for managing or delaying the evolution of ACB resistance to Bt corn plants.

Near-Isogenic Cry1F-Resistant Strain of Spodoptera frugiperda (Lepidoptera: Noctuidae) to Investigate Fitness Cost Associated With Resistance in Brazil.

PubMed

Horikoshi, Renato J; Bernardi, Oderlei; Bernardi, Daniel; Okuma, Daniela M; Farias, Juliano R; Miraldo, Leonardo L; Amaral, Fernando S A; Omoto, Celso

2016-04-01

Field-evolved resistance to Cry1F maize in Spodoptera frugiperda (J.E. Smith) populations in Brazil was reported in 2014. In this study, to investigate fitness costs, we constructed a near-isogenic S. frugiperda-resistant strain (R-Cry1F) using Cry1F-resistant and Cry1F-susceptible strains sharing a close genetic background. A near-isogenic R-Cry1F strain was obtained by eight repeated backcrossings, each followed by sib-mating and selection among resistant and susceptible strains. Fitness cost parameters were evaluated by comparing the biological performance of resistant, susceptible, and heterozygous strains on artificial diet. Fitness parameters monitored included development time and survival rates of egg, larval, pupal, and egg-to-adult periods; sex ratio; adult longevity; timing of preoviposition, oviposition, and postoviposition; fecundity; and fertility. A fertility life table was also calculated. The near-isogenic R-Cry1F strain showed lower survival rate of eggs (32%), when compared with Sus and reciprocal crosses (41 and 55%, respectively). The number of R-Cry1F insects that completed the life cycle was reduced to ∼25%, compared with the Sus strain with ∼32% reaching the adult stage. The mean generation time (T) of R-Cry1F strain was ∼2 d shorter than R-Cry1F♂×Sus♀ and Sus strains. The reproductive parameters of R-Cry1F strain were similar to the Sus strain. However, fewer females were produced by R-Cry1F strain than R-Cry1F♀×Sus♂ and more females than R-Cry1F♂×Sus♀. In summary, no relevant fitness costs are observed in a near-isogenic Cry1F-resistant strain of S. frugiperda, indicating stability of resistance to Cry1F protein in Brazilian populations of this species in the absence of selection pressure.

Bt Cry1Ie Toxin Does Not Impact the Survival and Pollen Consumption of Chinese Honey Bees, Apis cerana cerana (Hymenoptera, Apidae).

PubMed

Dai, Ping-Li; Jia, Hui-Ru; Jack, Cameron J; Geng, Li-Li; Liu, Feng; Hou, Chun-Sheng; Diao, Qing-Yun; Ellis, James D

2016-12-01

The cry1Ie gene may be a good candidate for the development of Bt maize because over-expression of Cry1Ie is highly toxic to Lepidopteran pests such as Heliothis armigera Hübner and Ostrinia furnacalis Guenée. The Bt cry1Ie gene also has no cross resistance with other insecticidal proteins such as Cry1Ab, Cry1Ac, Cry1Ah, or Cry1F. Chinese honey bees (Apis cerana cerana) are potentially exposed to insect-resistant genetically modified (IRGM) crops expressing Cry1Ie toxin via the collection of IRGM crop pollen. In this study, we tested whether Chinese honey bee workers are negatively affected by sugar syrup containing 20, 200, or 20,000 ng/ml Cry1Ie toxin and 48 ng/ml imidacloprid under controlled laboratory conditions. Our results demonstrated that the Cry1Ie toxin does not adversely impact survival and pollen consumption of Chinese honey bees. However, imidacloprid decreases Chinese honey bee survival and the total pollen consumption on the 5th, 6th, and 18th d of exposure. The described bioassay is suitable to assess the effects of GM expressed toxins against honey bee. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions*

PubMed Central

Yuan, Li; Seong, Eunju; Beuscher, James L.; Arikkath, Jyothi

2015-01-01

The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome. PMID:25724647

Nucleic acids encoding human trithorax protein

DOEpatents

Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

2001-01-01

In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

Persistence of Bt Bacillus thuringiensis Cry1Aa toxin in various soils determined by physicochemical reactions

NASA Astrophysics Data System (ADS)

Helassa, N.; Noinville, S.; Déjardin, P.; Janot, J. M.; Quiquampoix, H.; Staunton, S.

2009-04-01

Insecticidal Cry proteins from the soil bacterium, Bacillus thuringiensis (Bt) are produced by a class of genetically modified (GM) crops, and released into soils through root exudates and upon decomposition of residues. In contrast to the protoxin produced by the Bacillus, the protein produced in GM crops does not require activation in insect midguts and thereby potentially looses some of its species specificity. Although gene transfer and resistance emergence phenomena are well documented, the fate of these toxins in soil has not yet been clearly elucidated. Cry proteins, in common with other proteins, are adsorbed on soils and soil components. Adsorption on soil, and the reversibility of this adsorption is an important aspect of the environmental behaviour of these toxins. The orientation of the molecule and conformational changes on surfaces may modify the toxicity and confer some protection against microbial degradation. Adsorption will have important consequences for both the risk of exposition of non target species and the acquisition of resistance by target species. We have adopted different approaches to investigate the fate of Cry1Aa in soils and model minerals. In each series of experiments we endeavoured to maintain the protein in a monomeric form (pH above 6.5 and a high ionic strength imposed with 150 mM NaCl). The adsorption and the desorbability of the Cry1Aa Bt insecticidal protein were measured on two different homoionic clays: montmorillonite and kaolinite. Adsorption isotherms obtained followed a low affinity interaction for both clays and could be fitted using the Langmuir equation. Binding of the toxin decreased as the pH increased from 6.5 (close to the isoelectric point) to 9. Maximum adsorption was about 40 times greater on montmorillonite (1.71 g g-1) than on kaolinite (0.04 g g-1) in line with the contrasting respective specific surface areas of the minerals. Finally, some of the adsorbed toxin was desorbed by water and more, about 36 %, by high pH buffers, indicating that it was not extremely tightly bound. Moreover, the toxin was easily and quasi-completely desorbed using zwiterrionic and non-ionic detergents. We have compared the persistence of Cry1Aa on various soils over several weeks varying microbial activity (inhibition or activation). Neither physical nor chemical inhibition of microbial activity led to enhanced persistence of the protein in soil. Stimulation of microbial activity did not accelerate loss of detectable protein. These findings suggest that loss of protein in soil is not determined by microbial breakdown. Chemical fixation and conformational changes may contribute to the observed trends. Hydrophobic interactions with soil organo-mineral surfaces may play an important role in both the adsorption and subsequent changes in conformation of the protein.

Improvement and efficient display of Bacillus thuringiensis toxins on M13 phages and ribosomes.

PubMed

Pacheco, Sabino; Cantón, Emiliano; Zuñiga-Navarrete, Fernando; Pecorari, Frédéric; Bravo, Alejandra; Soberón, Mario

2015-12-01

Bacillus thuringiensis (Bt) produces insecticidal proteins that have been used worldwide in the control of insect-pests in crops and vectors of human diseases. However, different insect species are poorly controlled by the available Bt toxins or have evolved resistance to these toxins. Evolution of Bt toxicity could provide novel toxins to control insect pests. To this aim, efficient display systems to select toxins with increased binding to insect membranes or midgut proteins involved in toxicity are likely to be helpful. Here we describe two display systems, phage display and ribosome display, that allow the efficient display of two non-structurally related Bt toxins, Cry1Ac and Cyt1Aa. Improved display of Cry1Ac and Cyt1Aa on M13 phages was achieved by changing the commonly used peptide leader sequence of the coat pIII-fusion protein, that relies on the Sec translocation pathway, for a peptide leader sequence that relies on the signal recognition particle pathway (SRP) and by using a modified M13 helper phage (Phaberge) that has an amber mutation in its pIII genomic sequence and preferentially assembles using the pIII-fusion protein. Also, both Cry1Ac and Cyt1Aa were efficiently displayed on ribosomes, which could allow the construction of large libraries of variants. Furthermore, Cry1Ac or Cyt1Aa displayed on M13 phages or ribosomes were specifically selected from a mixture of both toxins depending on which antigen was immobilized for binding selection. These improved systems may allow the selection of Cry toxin variants with improved insecticidal activities that could counter insect resistances.

[Dynamics of Cry1ab protein content in the rhizosphere soil and straw debris of transgenic Bt corn].

PubMed

Li, Fan; Wang, Min; Sun, Hong-Wei; Yang, Shu-Ke; Lu, Xing-Bo

2013-07-01

By using ELISA test kits, a field investigation was conducted on the degradation dynamics of CrylAb protein in the rhizosphere soil of Bt corn MON810 at its different growth stages and in the MON810 straws returned into field after harvest. Three models (shift-log model, exponential model, and bi-exponential model) were used to fit the degradation dynamics of the Cry1 Ab protein from the straw debris, and the DT50 and DT90, values were estimated. There existed great differences in the CrylAb protein content in the rhizosphere soil of MON810 at its different growth stages, but overall, the CrylAb protein content was decreased remarkably with the growth of MON810. The degradation of Cry1 Ab protein from the straws covered on soil surface and buried in soil showed the same two-stage pattern, i.e., more rapid at early stage and slow-stable in later period. Within the first week after straw return, the degradation rate of the CrylAb protein from the straws covered on soil surface was significantly higher than that from the straws buried in soil. At 10 d, the degradation rate of the CrylAb protein from the straws covered on soil surface and buried in soil was basically the same, being 88.8% and 88.6%, respectively. After 20 days, the degradation of CrylAb protein entered slow-stable stage. Till at 180 d, a small amount of Cry1Ab protein could still be detected in the straw debris. All of the three models used in this study could fit the decay pattern of the CrylAb protein from the straw debris in field. By comparing the correlation coefficient (r) and the consistency between the measured and calculated DT90, bi-exponential model was considered to be the best.

Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock.

PubMed

Rubin, Elad B; Shemesh, Yair; Cohen, Mira; Elgavish, Sharona; Robertson, Hugh M; Bloch, Guy

2006-11-01

The circadian clock of the honey bee is implicated in ecologically relevant complex behaviors. These include time sensing, time-compensated sun-compass navigation, and social behaviors such as coordination of activity, dance language communication, and division of labor. The molecular underpinnings of the bee circadian clock are largely unknown. We show that clock gene structure and expression pattern in the honey bee are more similar to the mouse than to Drosophila. The honey bee genome does not encode an ortholog of Drosophila Timeless (Tim1), has only the mammalian type Cryptochrome (Cry-m), and has a single ortholog for each of the other canonical "clock genes." In foragers that typically have strong circadian rhythms, brain mRNA levels of amCry, but not amTim as in Drosophila, consistently oscillate with strong amplitude and a phase similar to amPeriod (amPer) under both light-dark and constant darkness illumination regimes. In contrast to Drosophila, the honey bee amCYC protein contains a transactivation domain and its brain transcript levels oscillate at virtually an anti-phase to amPer, as it does in the mouse. Phylogenetic analyses indicate that the basal insect lineage had both the mammalian and Drosophila types of Cry and Tim. Our results suggest that during evolution, Drosophila diverged from the ancestral insect clock and specialized in using a set of clock gene orthologs that was lost by both mammals and bees, which in turn converged and specialized in the other set. These findings illustrate a previously unappreciated diversity of insect clockwork and raise critical questions concerning the evolution and functional significance of species-specific variation in molecular clockwork.

Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

PubMed

Obata, Fumiaki; Tanaka, Shiho; Kashio, Soshiro; Tsujimura, Hidenobu; Sato, Ryoichi; Miura, Masayuki

2015-07-08

Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery. Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation. With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

Binding site concentration explains the differential susceptibility of Chilo suppressalis and Sesamia inferens to Cry1A-producing rice.

PubMed

Han, Lanzhi; Han, Chao; Liu, Zewen; Chen, Fajun; Jurat-Fuentes, Juan Luis; Hou, Maolin; Peng, Yufa

2014-08-01

Chilo suppressalis and Sesamia inferens are two important lepidopteran rice pests that occur concurrently during outbreaks in paddy fields in the main rice-growing areas of China. Previous and current field tests demonstrate that the transgenic rice line Huahui 1 (HH1) producing a Cry1Ab-Cry1Ac hybrid toxin from the bacterium Bacillus thuringiensis reduces egg and larval densities of C. suppressalis but not of S. inferens. This differential susceptibility to HH1 rice correlates with the reduced susceptibility to Cry1Ab and Cry1Ac toxins in S. inferens larvae compared to C. suppressalis larvae. The goal of this study was to identify the mechanism responsible for this differential susceptibility. In saturation binding assays, both Cry1Ab and Cry1Ac toxins bound with high affinity and in a saturable manner to midgut brush border membrane vesicles (BBMV) from C. suppressalis and S. inferens larvae. While binding affinities were similar, a dramatically lower concentration of Cry1A toxin binding sites was detected for S. inferens BBMV than for C. suppressalis BBMV. In contrast, no significant differences between species were detected for Cry1Ca toxin binding to BBMV. Ligand blotting detected BBMV proteins binding Cry1Ac or Cry1Ca toxins, some of them unique to C. suppressalis or S. inferens. These data support that reduced Cry1A binding site concentration is associated with a lower susceptibility to Cry1A toxins and HH1 rice in S. inferens larvae than in C. suppressalis larvae. Moreover, our data support Cry1Ca as a candidate for pyramiding efforts with Cry1A-producing rice to extend the activity range and durability of this technology against rice stem borers. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

PxAPN5 serves as a functional receptor of Cry2Ab in Plutella xylostella (L.) and its binding domain analysis.

PubMed

Pan, Zhi-Zhen; Xu, Lian; Liu, Bo; Zhang, Jing; Chen, Zheng; Chen, Qing-Xi; Zhu, Yu-Jing

2017-12-01

Lepidopteran midgut aminopeptidases N (APNs) are widely studied for their potential roles as one of the receptors for Bacillus thuringiensis (Bt) crystal toxins. In the present study, a loss of function analyses by RNAi (RNA interference) silencing of the Plutella xylostella APN5 (PxAPN5), a binding protein of Bt crystal toxin Cry2Ab, were performed. The knocking down of PxAPN5 in P. xylostella larvae greatly reduced their susceptibility to Cry2Ab and led to a decrease of Cry2Ab binding to P. xylostella midgut. Four truncated fragments of PxAPN5 were further constructed and expressed in Escherichia coli (E.coli) to find the binding region of PxAPN5 to Cry2Ab. The ligand blot result indicated that D1 domain (residues 1-262) and D3 domain (residues 510-620) of PxAPN5 could specially bind to Cry2Ab. Copyright © 2017 Elsevier B.V. All rights reserved.

Binding of Substrate Locks the Electrochemistry of CRY-DASH into DNA Repair.

PubMed

Gindt, Yvonne M; Messyasz, Adriana; Jumbo, Pamela I

2015-05-12

VcCry1, a member of the CRY-DASH family, may serve two diverse roles in vivo, including blue-light signaling and repair of UV-damaged DNA. We have discovered that the electrochemistry of the flavin adenine dinucleotide cofactor of VcCry1 is locked to cycle only between the hydroquinone and neutral semiquinone states when UV-damaged DNA is present. Other potential substrates, including undamaged DNA and ATP, have no discernible effect on the electrochemistry, and the kinetics of the reduction is unaffected by damaged DNA. Binding of the damaged DNA substrate determines the role of the protein and prevents the presumed photochemistry required for blue-light signaling.

A method for in Vivo radiolabeling Bacillus thuringiensis native δ-endotoxin crystals

Treesearch

C. Noah Koller; Leah S. Bauer; Robert M. Hollingworth

1995-01-01

The entomocidal CryIIIA δ-endotoxin protein of Bacillus thuringiensis var. tenebrionis is distinctive in chemistry and host range. In contrast to other δ-endotoxins, the CryIIIA parasporal crystals are toxic within the acidic midgut environment of several coleopteran species, particularly those in the family...

BCL11B is frequently downregulated in HTLV-1-infected T-cells through Tax-mediated proteasomal degradation.

PubMed

Permatasari, Happy Kurnia; Nakahata, Shingo; Ichikawa, Tomonaga; Morishita, Kazuhiro

2017-08-26

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia-lymphoma (ATLL). The HTLV-1-encoded protein Tax plays important roles in the proliferation of HTLV-1-infected T-cells by affecting cellular proteins. In this study, we showed that Tax transcriptionally and post-transcriptionally downregulates the expression of the tumor suppressor gene B-cell leukemia/lymphoma 11B (BCL11B), which encodes a lymphoid-related transcription factor. BCL11B expression was downregulated in HTLV-1-infected T-cell lines at the mRNA and protein levels, and forced expression of BCL11B suppressed the proliferation of these cells. The proteasomal inhibitor MG132 increased BCL11B expression in HTLV-1-infected cell lines, and colocalization of Tax with BCL11B was detected in the cytoplasm of HTLV-1-infected T-cells following MG132 treatment. shRNA knock-down of Tax expression also increased the expression of BCL11B in HTLV-1-infected cells. Moreover, we found that Tax physically binds to BCL11B protein and induces the polyubiquitination of BCL11B and proteasome-dependent degradation of BCL11B. Thus, inactivation of BCL11B by Tax protein may play an important role in the Tax-mediated leukemogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

NASA Astrophysics Data System (ADS)

Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

2016-01-01

We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.

Isotope-encoded Carboxyl Group Footprinting for Mass Spectrometry-based Protein Conformational Studies

PubMed Central

Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

2015-01-01

We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded “heavy” and “light” GEE are used to label separately the two states of the Orange Carotenoid Protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the “heavy” and “light” peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting. PMID:26384685

Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

DOE PAGES

Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; ...

2015-09-18

Here, we report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded “heavy” and “light” GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the “heavy”more » and “light” peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.« less

Knockout of a P-glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua.

PubMed

Zuo, Y-Y; Huang, J-L; Wang, J; Feng, Y; Han, T-T; Wu, Y-D; Yang, Y-H

2018-02-01

P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta-cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua. © 2017 The Royal Entomological Society.

A laboratory assessment of the potential effect of Cry1Ab/Cry2Aj-containing Bt maize pollen on Folsomia candida by toxicological and biochemical analyses.

PubMed

Zhang, Bing; Yang, Yan; Zhou, Xiang; Shen, Ping; Peng, Yufa; Li, Yunhe

2017-03-01

The common soil arthropod Folsomia candida can survive well when fed only maize pollen and thus may be exposed to insecticidal proteins by ingesting insect-resistant genetically engineered maize pollen containing Bacillus thuringiensis (Bt) proteins when being released into the soil. Laboratory experiments were conducted to assess the potential effects of Cry1Ab/Cry2Aj-producing transgenic Bt maize (Shuangkang 12-5) pollen on F. candida fitness. Survival, development, and the reproduction were not significantly reduced when F. candida fed on Bt maize pollen rather than on non-Bt maize pollen, but these parameters were significantly reduced when F. candida fed on non-Bt maize pollen containing the protease inhibitor E-64 at 75 μg/g pollen. The intrinsic rate of increase (r m ) was not significantly reduced when F. candida fed on Bt maize pollen but was significantly reduced when F. candida fed on non-Bt maize pollen containing E-64. The activities of antioxidant-related enzymes in F. candida were not significantly affected when F. candida fed on Bt maize pollen but were significantly increased when F. candida fed on non-Bt pollen containing E-64. The results demonstrate that consumption of Bt maize pollen containing Cry1Ab/Cry2Aj has no lethal or sublethal effects on F. candida. Copyright © 2017 Elsevier Ltd. All rights reserved.

Changes in active site histidine hydrogen bonding trigger cryptochrome activation

PubMed Central

Ganguly, Abir; Manahan, Craig C.; Top, Deniz; Yee, Estella F.; Lin, Changfan; Young, Michael W.; Thiel, Walter; Crane, Brian R.

2016-01-01

Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa. Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

The Flavoproteins CryD and VvdA Cooperate with the White Collar Protein WcoA in the Control of Photocarotenogenesis in Fusarium fujikuroi

PubMed Central

Castrillo, Marta; Avalos, Javier

2015-01-01

Light stimulates carotenoid biosynthesis in the ascomycete fungus Fusarium fujikuroi through transcriptional activation of the structural genes of the pathway carRA, carB, and cart, but the molecular basis of this photoresponse is unknown. The F. fujikuroi genome contains genes for different predicted photoreceptors, including the WC protein WcoA, the DASH cryptochrome CryD and the Vivid-like flavoprotein VvdA. We formerly found that null mutants of wcoA, cryD or vvdA exhibit carotenoid photoinduction under continuous illumination. Here we show that the wild type exhibits a biphasic response in light induction kinetics experiments, with a rapid increase in carotenoid content in the first hours, a transient arrest and a subsequent slower increase. The mutants of the three photoreceptors show different kinetic responses: the wcoA mutants are defective in the rapid response, the cryD mutants are affected in the slower response, while the fast and slow responses were respectively enhanced and attenuated in the vvdA mutants. Transcriptional analyses of the car genes revealed a strong reduction of dark and light-induced transcript levels in the wcoA mutants, while minor or no reductions were found in the cryD mutants. Formerly, we found no change on carRA and carB photoinduction in vvdA mutants. Taken together, our data suggest a cooperative participation of WcoA and CryD in early and late stages of photoinduction of carotenoid biosynthesis in F. fujikuroi, and a possible modulation of WcoA activity by VvdA. An unexpected transcriptional induction by red light of vvdA, cryD and carRA genes suggest the participation of an additional red light-absorbing photoreceptor. PMID:25774802

An overview of the safety and biological effects of Bacillus thuringiensis Cry toxins in mammals.

PubMed

Rubio-Infante, Néstor; Moreno-Fierros, Leticia

2016-05-01

Crystal proteins (Cry) produced during the growth and sporulation phases of Bacillus thuringiensis (Bt) bacterium are known as delta endotoxins. These toxins are being used worldwide as bioinsecticides to control pests in agriculture, and some Cry toxins are used against mosquitoes to control vector transmission. This review summarizes the relevant information currently available regarding the biosafety and biological effects that Bt and its insecticidal Cry proteins elicit in mammals. This work was performed because of concerns regarding the possible health impact of Cry toxins on vertebrates, particularly because Bt toxins might be associated with immune-activating or allergic responses. The controversial data published to date are discussed in this review considering earlier toxicological studies of B. thuringiensis, spores, toxins and Bt crops. We discussed the experimental studies performed in humans, mice, rats and sheep as well as in diverse mammalian cell lines. Although the term 'toxic' is not appropriate for defining the effects these toxins have on mammals, they cannot be considered innocuous, as they have some physiological effects that may become pathological; thus, trials that are more comprehensive are necessary to determine their effects on mammals because knowledge in this field remains limited. Copyright © 2015 John Wiley & Sons, Ltd.

Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.

PubMed

Pardo-López, Liliana; Soberón, Mario; Bravo, Alejandra

2013-01-01

Bacillus thuringiensis bacteria are insect pathogens that produce different Cry and Cyt toxins to kill their hosts. Here we review the group of three-domain Cry (3d-Cry) toxins. Expression of these 3d-Cry toxins in transgenic crops has contributed to efficient control of insect pests and a reduction in the use of chemical insecticides. The mode of action of 3d-Cry toxins involves sequential interactions with several insect midgut proteins that facilitate the formation of an oligomeric structure and induce its insertion into the membrane, forming a pore that kills midgut cells. We review recent progress in our understanding of the mechanism of action of these Cry toxins and focus our attention on the different mechanisms of resistance that insects have evolved to counter their action, such as mutations in cadherin, APN and ABC transporter genes. Activity of Cry1AMod toxins, which are able to form toxin oligomers in the absence of receptors, against different resistant populations, including those affected in the ABC transporter and the role of dominant negative mutants as antitoxins, supports the hypothesis that toxin oligomerization is a limiting step in the Cry insecticidal activity. Knowledge of the action of 3d-Cry toxin and the resistance mechanisms to these toxins will set the basis for a rational design of novel toxins to overcome insect resistance, extending the useful lifespan of Cry toxins in insect control programs. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Increased toxicity of Bacillus thuringiensis Cry3Aa against Crioceris quatuordecimpunctata, Phaedon brassicae and Colaphellus bowringi by a Tenebrio molitor cadherin fragment.

PubMed

Gao, Yulin; Jurat-Fuentes, Juan Luis; Oppert, Brenda; Fabrick, Jeffrey A; Liu, Chenxi; Gao, Jianhua; Lei, Zhongren

2011-09-01

Biopesticides containing Cry insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are effective against many lepidopteran pests, but there is a lack of Bt-based pesticides for efficient control of important coleopteran pests. Based on the reported increase in Bt toxin oligomerization by a polypeptide from the Cry3Aa receptor cadherin in Tenebrio molitor (Coleoptera: Tenebrionidae), it was hypothesized that this cadherin peptide, rTmCad1p, would enhance Cry3Aa toxicity towards coleopteran larvae. To test this hypothesis, the relative toxicity of Cry3Aa, with or without rTmCad1p, against damaging chrysomelid vegetable pests of China was evaluated. Cry3Aa toxicity was evaluated in the spotted asparagus beetle (Crioceris quatuordecimpunctata), cabbage leaf beetle (Colaphellus bowringi) and daikon leaf beetle (Phaedon brassicae). To assess the effect of rTmCad1p on Cry3Aa toxicity, neonate larvae were fed Cry3Aa toxin alone or in combination with increasing amounts of rTmCad1p. The data demonstrated that Cry3Aa toxicity was significantly increased in all three vegetable pests, resulting in as much as a 15.3-fold increase in larval mortality. The application of rTmCad1p to enhance Cry3Aa insecticidal activity has potential for use in increasing range and activity levels against coleopteran pests displaying low susceptibility to Bt-based biopesticides. Copyright © 2011 Society of Chemical Industry.

Melting Lizards and Crying Mailboxes: Children's Preferential Recall of Minimally Counterintuitive Concepts

ERIC Educational Resources Information Center

Banerjee, Konika; Haque, Omar S.; Spelke, Elizabeth S.

2013-01-01

Previous research with adults suggests that a catalog of minimally counterintuitive concepts, which underlies supernatural or religious concepts, may constitute a cognitive optimum and is therefore cognitively encoded and culturally transmitted more successfully than either entirely intuitive concepts or maximally counterintuitive concepts. This…

FIN219/JAR1 and cryptochrome1 antagonize each other to modulate photomorphogenesis under blue light in Arabidopsis

PubMed Central

2018-01-01

Plant development is affected by the integration of light and phytohormones, including jasmonates (JAs). To address the molecular mechanisms of possible interactions between blue light and JA signaling in Arabidopsis thaliana, we used molecular and transgenic approaches to understand the regulatory relationships between FAR-RED INSENSITIVE 219 (FIN219)/JASMONATE RESISTANT1 (JAR1) and the blue-light photoreceptor cryptochrome1 (CRY1). FIN219 overexpression in the wild type resulted in a short-hypocotyl phenotype under blue light. However, FIN219 overexpression in cry1, cry2 and cry1cry2 double mutant backgrounds resulted in phenotypes similar to their respective mutant backgrounds, which suggests that FIN219 function may require blue light photoreceptors. Intriguingly, FIN219 overexpression in transgenic plants harboring ectopic expression of the C terminus of CRY1 (GUS-CCT1), which exhibits a hypersensitive short-hypocotyl phenotype in all light conditions including darkness, led to a rescued phenotype under all light conditions except red light. Further expression studies showed mutual suppression between FIN219 and CRY1 under blue light. Strikingly, FIN219 overexpression in GUS-CCT1 transgenic lines (FIN219-OE/GUS-CCT1) abolished GUS-CCT1 fusion protein under blue light, whereas GUS-CCT1 fusion protein was stable in the fin219-2 mutant background (fin219-2/GUS-CCT1). Moreover, FIN219 strongly interacted with COP1 under blue light, and methyl JA (MeJA) treatment enhanced the interaction between FIN219 and GUS-CCT1 under blue light. Furthermore, FIN219 level affected GUS-CCT1 seedling responses such as anthocyanin accumulation and bacterial resistance under various light conditions and MeJA treatment. Thus, FIN219/JAR1 and CRY1 antagonize each other to modulate photomorphogenic development of seedlings and stress responses in Arabidopsis. PMID:29561841

FIN219/JAR1 and cryptochrome1 antagonize each other to modulate photomorphogenesis under blue light in Arabidopsis.

PubMed

Chen, Huai-Ju; Fu, Tsu-Yu; Yang, Shao-Li; Hsieh, Hsu-Liang

2018-03-01

Plant development is affected by the integration of light and phytohormones, including jasmonates (JAs). To address the molecular mechanisms of possible interactions between blue light and JA signaling in Arabidopsis thaliana, we used molecular and transgenic approaches to understand the regulatory relationships between FAR-RED INSENSITIVE 219 (FIN219)/JASMONATE RESISTANT1 (JAR1) and the blue-light photoreceptor cryptochrome1 (CRY1). FIN219 overexpression in the wild type resulted in a short-hypocotyl phenotype under blue light. However, FIN219 overexpression in cry1, cry2 and cry1cry2 double mutant backgrounds resulted in phenotypes similar to their respective mutant backgrounds, which suggests that FIN219 function may require blue light photoreceptors. Intriguingly, FIN219 overexpression in transgenic plants harboring ectopic expression of the C terminus of CRY1 (GUS-CCT1), which exhibits a hypersensitive short-hypocotyl phenotype in all light conditions including darkness, led to a rescued phenotype under all light conditions except red light. Further expression studies showed mutual suppression between FIN219 and CRY1 under blue light. Strikingly, FIN219 overexpression in GUS-CCT1 transgenic lines (FIN219-OE/GUS-CCT1) abolished GUS-CCT1 fusion protein under blue light, whereas GUS-CCT1 fusion protein was stable in the fin219-2 mutant background (fin219-2/GUS-CCT1). Moreover, FIN219 strongly interacted with COP1 under blue light, and methyl JA (MeJA) treatment enhanced the interaction between FIN219 and GUS-CCT1 under blue light. Furthermore, FIN219 level affected GUS-CCT1 seedling responses such as anthocyanin accumulation and bacterial resistance under various light conditions and MeJA treatment. Thus, FIN219/JAR1 and CRY1 antagonize each other to modulate photomorphogenic development of seedlings and stress responses in Arabidopsis.

Field‐evolved resistance to Bt maize in sugarcane borer (Diatraea saccharalis) in Argentina

PubMed Central

Grimi, Damián A; Ramos, María Laura; Machado, Marcos; Ocampo, Federico; Willse, Alan; Martinelli, Samuel; Head, Graham

2018-01-01

Abstract BACKGROUND Maize technologies expressing Bacillus thuringiensis (Bt) insecticidal proteins are widely used in Argentina to control sugarcane borer (Diatraea saccharalis Fabricius). Unexpected D. saccharalis damage was observed to Bt maize events TC1507 (expressing Cry1F) and MON 89034 × MON 88017 (expressing Cry1A.105 and Cry2Ab2) in an isolated area of San Luis Province. Diatraea saccharalis larvae were sampled from MON 89034 × MON 88017 fields in the area to generate a resistant strain (RR), which was subsequently characterized in plant and diet bioassays. RESULTS Survivorship of the RR strain was high on TC1507 leaf tissue, intermediate on MON 89034 × MON 88017, and low on MON 810 (expressing Cry1Ab). The RR strain had high resistance to Cry1A.105 (186.74‐fold) and no resistance to Cry2Ab2 in diet bioassays. These results indicate resistance to Cry1F and Cry1A.105 (and likely cross‐resistance between them) but not to Cry1Ab or Cry2Ab2. Resistance to MON 89034 × MON 88017 was functionally recessive. Reviews of grower records suggest that resistance initially evolved to Cry1F, conferring cross‐resistance to Cry1A.105, with low refuge compliance as the primary cause. A mitigation plan was implemented in San Luis that included technology rotation, field monitoring, and grower education on best management practices (BMPs) including refuges. CONCLUSION In the affected area, the resistance to Cry1F and Cry1A.105 is being managed effectively through use of MON 89034 × MON 88017 and MON 810 in combination with BMPs, and no spread of resistance to other regions has been observed. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:29095565

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

PubMed Central

Kasman, Laura M.; Lukowiak, Andrew A.; Garczynski, Stephen F.; McNall, Rebecca J.; Youngman, Phil; Adang, Michael J.

1998-01-01

Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning. PMID:9687463

A Meta-Analysis of Effects of Bt Crops on Honey Bees (Hymenoptera: Apidae)

PubMed Central

Duan, Jian J.; Marvier, Michelle; Huesing, Joseph; Dively, Galen; Huang, Zachary Y.

2008-01-01

Background Honey bees (Apis mellifera L.) are the most important pollinators of many agricultural crops worldwide and are a key test species used in the tiered safety assessment of genetically engineered insect-resistant crops. There is concern that widespread planting of these transgenic crops could harm honey bee populations. Methodology/Principal Findings We conducted a meta-analysis of 25 studies that independently assessed potential effects of Bt Cry proteins on honey bee survival (or mortality). Our results show that Bt Cry proteins used in genetically modified crops commercialized for control of lepidopteran and coleopteran pests do not negatively affect the survival of either honey bee larvae or adults in laboratory settings. Conclusions/Significance Although the additional stresses that honey bees face in the field could, in principle, modify their susceptibility to Cry proteins or lead to indirect effects, our findings support safety assessments that have not detected any direct negative effects of Bt crops for this vital insect pollinator. PMID:18183296

Variation in microRNA levels between Cry1Ab-resistant and susceptible strains of the European corn borer, Ostrinia nubilalis (Hübner)

USDA-ARS?s Scientific Manuscript database

Transgenic maize hybrids that express insecticidal Bacillus thuringiensis (Bt) crystalline (Cry) protein toxins effectively protect against feeding damage inflicted by the insect pest the European corn borer, Ostrinia nubilalis. Field monitoring and laboratory selections have detected varying level...

Comparison of fumonisin contamination using HPLC and ELISA methods in bt and near-isogenic maize hybrids infested with European corn borer or western bean cutworm.

PubMed

Bowers, Erin; Hellmich, Richard; Munkvold, Gary

2014-07-09

Field trials were conducted from 2007 to 2010 to compare grain fumonisin levels among non-Bt maize hybrids and Bt hybrids with transgenic protection against manual infestations of European corn borer (ECB) and Western bean cutworm (WBC). HPLC and ELISA were used to measure fumonisin levels. Results of the methods were highly correlated, but ELISA estimates were higher. Bt hybrids experienced less insect injury, Fusarium ear rot, and fumonisin contamination compared to non-Bt hybrids. WBC infestation increased fumonisin content compared to natural infestation in non-Bt and hybrids expressing Cry1Ab protein in five of eight possible comparisons; in Cry1F hybrids, WBC did not impact fumonisins. These results indicate that WBC is capable of increasing fumonisin levels in maize. Under WBC infestation, Cry1F mitigated this risk more consistently than Cry1Ab or non-Bt hybrids. Transgenically expressed Bt proteins active against multiple lepidopteran pests can provide broad, consistent reductions in the risk of fumonisin contamination.

I thought that I heard you laughing: Contextual facial expressions modulate the perception of authentic laughter and crying.

PubMed

Lavan, Nadine; Lima, César F; Harvey, Hannah; Scott, Sophie K; McGettigan, Carolyn

2015-01-01

It is well established that categorising the emotional content of facial expressions may differ depending on contextual information. Whether this malleability is observed in the auditory domain and in genuine emotion expressions is poorly explored. We examined the perception of authentic laughter and crying in the context of happy, neutral and sad facial expressions. Participants rated the vocalisations on separate unipolar scales of happiness and sadness and on arousal. Although they were instructed to focus exclusively on the vocalisations, consistent context effects were found: For both laughter and crying, emotion judgements were shifted towards the information expressed by the face. These modulations were independent of response latencies and were larger for more emotionally ambiguous vocalisations. No effects of context were found for arousal ratings. These findings suggest that the automatic encoding of contextual information during emotion perception generalises across modalities, to purely non-verbal vocalisations, and is not confined to acted expressions.

pH-Controlled Bacillus thuringiensis Cry1Ac Protoxin Loading and Release from Polyelectrolyte Microcapsules

PubMed Central

Yang, Wenhui; He, Kanglai; Zhang, Jie; Guo, Shuyuan

2012-01-01

Crystal proteins synthesized by Bacillus thuringiensis (Bt) have been used as biopesticides because of their toxicity to the insect larval hosts. To protect the proteins from environmental stress to extend their activity, we have developed a new microcapsule formulation. Poly (acrylic acid) (PAH) and poly (styrene sulfonate) (PSS) were fabricated through layer-by-layer self-assembly based on a CaCO3 core. Cry1Ac protoxins were loaded into microcapsules through layer-by-layer self-assembly at low pH, and the encapsulated product was stored in water at 4°C. Scanning electron microscopy (SEM) was used to observe the morphology of the capsules. To confirm the successful encapsulation, the loading results were observed with a confocal laser scattering microscope (CLSM), using fluorescein-labeled Cry1Ac protoxin (FITC-Cry1Ac). The protoxins were released from the capsule under the alkaline condition corresponding to the midgut of certain insects, a condition which seldom exists elsewhere in the environment. The following bioassay experiment demonstrated that the microcapsules with Cry1Ac protoxins displayed approximately equivalent insecticidal activity to the Asian corn borer compared with free Cry1Ac protoxins, and empty capsules proved to have no effect on insects. Further result also indicated that the formulation could keep stable under the condition of heat and desiccation. These results suggest that this formulation provides a promising methodology that protects protoxins from the environment and releases them specifically in the target insects’ midgut, which has shown potential as biopesticide in the field. PMID:23024810

Cry1Ac production is costly for native plants attacked by non-Cry1Ac-targeted herbivores in the field.

PubMed

McGale, Erica; Diezel, Celia; Schuman, Meredith C; Baldwin, Ian T

2018-05-13

Plants are the primary producers in most terrestrial ecosystems and have complex defense systems to protect their produce. Defense-deficient, high-yielding agricultural monocultures attract abundant nonhuman consumers, but are alternatively defended through pesticide application and genetic engineering to produce insecticidal proteins such as Cry1Ac (Bacillus thuringiensis). These approaches alter the balance between yield protection and maximization but have been poorly contextualized to known yield-defense trade-offs in wild plants. The native plant Nicotiana attenuata was used to compare yield benefits of plants transformed to be defenseless to those with a full suite of naturally evolved defenses, or additionally transformed to ectopically produce Cry1Ac. An insecticide treatment allowed us to examine yield under different herbivore loads in N. attenuata's native habitat. Cry1Ac, herbivore damage, and growth parameters were monitored throughout the season. Biomass and reproductive correlates were measured at season end. Non-Cry1Ac-targeted herbivores dominated on noninsecticide-treated plants, and increased the yield drag of Cry1Ac-producing plants in comparison with endogenously defended or undefended plants. Insecticide-sprayed Cry1Ac-producing plants lagged less in stalk height, shoot biomass, and flower production. In direct comparison with the endogenous defenses of a native plant, Cry1Ac production did not provide yield benefits for plants under observed herbivore loads in a field study. © 2018 The Authors New Phytologist © 2018 New Phytologist Trust.

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

PubMed

Dong, Sa; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Xie, Yajing; Zhong, Jianfeng; Xu, Chongxin; Liu, Xianjin

2017-03-01

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D ) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10 -8  M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL -1 , respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL -1 ), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Comparison of compensatory reserve during lower-body negative pressure and hemorrhage in nonhuman primates.

PubMed

Hinojosa-Laborde, Carmen; Howard, Jeffrey T; Mulligan, Jane; Grudic, Greg Z; Convertino, Victor A

2016-06-01

Compensatory reserve was measured in baboons (n = 13) during hemorrhage (Hem) and lower-body negative pressure (LBNP) using a machine-learning algorithm developed to estimate compensatory reserve by detecting reductions in central blood volume during LBNP. The algorithm calculates compensatory reserve index (CRI) from normovolemia (CRI = 1) to cardiovascular decompensation (CRI = 0). The hypothesis was that Hem and LBNP will elicit similar CRI values and that CRI would have higher specificity than stroke volume (SV) in predicting decompensation. Blood was removed in four steps: 6.25%, 12.5%, 18.75%, and 25% of total blood volume. Four weeks after Hem, the same animals were subjected to four levels of LBNP that was matched on the basis of their central venous pressure. Data (mean ± 95% confidence interval) indicate that CRI decreased (P < 0.001) from baseline during Hem (0.69 ± 0.10, 0.57 ± 0.09, 0.36 ± 0.10, 0.16 ± 0.08, and 0.08 ± 0.03) and LBNP (0.76 ± 0.05, 0.66 ± 0.08, 0.36 ± 0.13, 0.23 ± 0.11, and 0.14 ± 0.09). CRI was not different between Hem and LBNP (P = 0.20). Linear regression analysis between Hem CRI and LBNP CRI revealed a slope of 1.03 and a correlation coefficient of 0.96. CRI exhibited greater specificity than SV in both Hem (92.3 vs. 82.1) and LBNP (94.8 vs. 83.1) and greater ROC AUC in Hem (0.94 vs. 0.84) and LBNP (0.94 vs. 0.92). These data support the hypothesis that Hem and LBNP elicited the same CRI response, suggesting that measurement of compensatory reserve is superior to SV as a predictor of cardiovascular decompensation.

Comparison of compensatory reserve during lower-body negative pressure and hemorrhage in nonhuman primates

PubMed Central

Howard, Jeffrey T.; Mulligan, Jane; Grudic, Greg Z.; Convertino, Victor A.

2016-01-01

Compensatory reserve was measured in baboons (n = 13) during hemorrhage (Hem) and lower-body negative pressure (LBNP) using a machine-learning algorithm developed to estimate compensatory reserve by detecting reductions in central blood volume during LBNP. The algorithm calculates compensatory reserve index (CRI) from normovolemia (CRI = 1) to cardiovascular decompensation (CRI = 0). The hypothesis was that Hem and LBNP will elicit similar CRI values and that CRI would have higher specificity than stroke volume (SV) in predicting decompensation. Blood was removed in four steps: 6.25%, 12.5%, 18.75%, and 25% of total blood volume. Four weeks after Hem, the same animals were subjected to four levels of LBNP that was matched on the basis of their central venous pressure. Data (mean ± 95% confidence interval) indicate that CRI decreased (P < 0.001) from baseline during Hem (0.69 ± 0.10, 0.57 ± 0.09, 0.36 ± 0.10, 0.16 ± 0.08, and 0.08 ± 0.03) and LBNP (0.76 ± 0.05, 0.66 ± 0.08, 0.36 ± 0.13, 0.23 ± 0.11, and 0.14 ± 0.09). CRI was not different between Hem and LBNP (P = 0.20). Linear regression analysis between Hem CRI and LBNP CRI revealed a slope of 1.03 and a correlation coefficient of 0.96. CRI exhibited greater specificity than SV in both Hem (92.3 vs. 82.1) and LBNP (94.8 vs. 83.1) and greater ROC AUC in Hem (0.94 vs. 0.84) and LBNP (0.94 vs. 0.92). These data support the hypothesis that Hem and LBNP elicited the same CRI response, suggesting that measurement of compensatory reserve is superior to SV as a predictor of cardiovascular decompensation PMID:27030667

Isolation of transcripts from Diabrotica virgifera virgifera LeConte responsive to the Bacillus thuringiensis toxin Cry3Bb1

EPA Science Inventory

Crystal proteins derived from Bacillus thuringiensis (Bt) have been widely used as a method of insect pest management for several decades. In recent years, a transgenic corn expressing the Cry3Bb1 toxin has been successfully used for protection against corn rootworm larvae (Genus...

Increased toxicity of Bacillus thuringiensis Cry3Aa against Crioceris quatuordecimpunctata, Phaedon brassicae and Colaphellus bowringi by a Tenebrio molitor cadherin fragment

USDA-ARS?s Scientific Manuscript database

BACKGROUND: Biopesticides containing Cry insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are effective against many lepidopteran pests, but there is a lack of Bt-based pesticides to efficiently control important coleopteran pests. Based on the reported increase of Bt toxin olig...

Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts

USDA-ARS?s Scientific Manuscript database

The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the coleopteran-specific Cry3Aa toxin from Bacillus thuringiensis (Bt). Larvae digest protein initially with cysteine peptidases in the anterior midgut and further with serine peptidases in middle and poste...

Update on monitoring of resistance to Bt cotton in key lepidopteran pests in the USA

USDA-ARS?s Scientific Manuscript database

Producers sprayed more Bollgard II to control target lepidopteran pests in 2010 than in previous years, and therefore concerns have been expressed that the susceptibility of the target lepidopteran pests to the Bt Cry1Ac and Cry2Ab proteins in Bollgard II has significantly decreased. However, resist...

Stable integration and expression of a cry1Ia gene conferring resistance to fall armyworm and boll weevil in cotton plants.

PubMed

Silva, Carliane Rc; Monnerat, Rose; Lima, Liziane M; Martins, Érica S; Melo Filho, Péricles A; Pinheiro, Morganna Pn; Santos, Roseane C

2016-08-01

Boll weevil is a serious pest of cotton crop. Effective control involves applications of chemical insecticides, increasing the cost of production and environmental pollution. The current genetically modified Bt crops have allowed great benefits to farmers but show activity limited to lepidopteran pests. This work reports on procedures adopted for integration and expression of a cry transgene conferring resistance to boll weevil and fall armyworm by using molecular tools. Four Brazilian cotton cultivars were microinjected with a minimal linear cassette generating 1248 putative lines. Complete gene integration was found in only one line (T0-34) containing one copy of cry1Ia detected by Southern blot. Protein was expressed in high concentration at 45 days after emergence (dae), decreasing by approximately 50% at 90 dae. Toxicity of the cry protein was demonstrated in feeding bioassays revealing 56.7% mortality to boll weevil fed buds and 88.1% mortality to fall armyworm fed leaves. A binding of cry1Ia antibody was found in the midgut of boll weevils fed on T0-34 buds in an immunodetection assay. The gene introduced into plants confers resistance to boll weevil and fall armyworm. Transmission of the transgene occurred normally to T1 progeny. All plants showed phenotypically normal growth, with fertile flowers and abundant seeds. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Dominant resistance to Bt cotton and minor cross-resistance to Bt toxin Cry2Ab in cotton bollworm from China

PubMed Central

Jin, Lin; Wei, Yiyun; Zhang, Lei; Yang, Yihua; Tabashnik, Bruce E; Wu, Yidong

2013-01-01

Evolution of resistance by insect pests threatens the long-term benefits of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Previous work has detected increases in the frequency of resistance to Bt toxin Cry1Ac in populations of cotton bollworm, Helicoverpa armigera, from northern China where Bt cotton producing Cry1Ac has been grown extensively for more than a decade. Confirming that trend, we report evidence from 2011 showing that the percentage of individuals resistant to a diagnostic concentration of Cry1Ac was significantly higher in two populations from different provinces of northern China (1.4% and 2.3%) compared with previously tested susceptible field populations (0%). We isolated two resistant strains: one from each of the two field-selected populations. Relative to a susceptible strain, the two strains had 460- and 1200-fold resistance to Cry1Ac, respectively. Both strains had dominant resistance to a diagnostic concentration of Cry1Ac in diet and to Bt cotton leaves containing Cry1Ac. Both strains had low, but significant cross-resistance to Cry2Ab (4.2- and 5.9-fold), which is used widely as the second toxin in two-toxin Bt cotton. Compared with resistance in other strains of H. armigera, the resistance in the two strains characterized here may be especially difficult to suppress. PMID:24478804

A multi-year field study to evaluate the environmental fate and agronomic effects of insecticide mixtures.

PubMed

Whiting, Sara A; Strain, Katherine E; Campbell, Laura A; Young, Bryan G; Lydy, Michael J

2014-11-01

A mixture of insecticides used in corn production was monitored over a three-year period in a field study to determine how long each persists in the environment, where each insecticide travels within the corn field, and the efficacy of using soil-applied insecticides with genetically modified corn. The genetically modified corn contained the insecticidal Cry1Ab and Cry3Bb1 proteins (Bt corn) and the Cry1Ab protein was found to persist only during the corn growing season in soil, runoff water, and runoff sediment with highest concentrations measured during pollination. Very low concentrations of Cry1Ab proteins were measured in soil collected in the non-Bt corn field, and no Cry1Ab proteins were detected in shallow groundwater or soil pore water. Clothianidin, a neonicotinoid insecticide used as a seed coating, was detected in all matrices and remained persistent throughout the year in soil pore water. Tefluthrin, a pyrethroid insecticide applied at planting to control corn rootworm larvae (Diabrotica spp., Coleoptera: Chrysomelidae) populations, was consistently detected in soil, runoff water, and runoff sediment during the corn growing season, but was not detected in groundwater or soil pore water. Tefluthrin did not have an effect on root damage from corn rootworm larvae feeding to Bt corn, but did prevent damage to non-Bt corn. A slight reduction in grain yield was observed in the non-Bt, no tefluthrin treatment when compared to all other treatments, but no significant difference in grain yield was observed among Bt corn treatments regardless of soil insecticide application. In the current study, the use of tefluthrin on Bt corn did not significantly affect crop damage or yield, and tefluthrin may travel off-site in runoff water and sediment. Copyright © 2014 Elsevier B.V. All rights reserved.

δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions.

PubMed

Yuan, Li; Seong, Eunju; Beuscher, James L; Arikkath, Jyothi

2015-04-24

The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Video imaging analysis of the plasma membrane permeabilizing effects of Bacillus thuringiensis insecticidal toxins in Sf9 cells.

PubMed

Villalon, M; Vachon, V; Brousseau, R; Schwartz, J L; Laprade, R

1998-01-05

The size and ionic selectivity of the pores formed by the insecticidal crystal protein Cry1C from Bacillus thuringiensis in the plasma membrane of Sf9 cells, an established cell line derived from the fall armyworm Spodoptera frugiperda, were analyzed with a video imaging technique. Changes in the permeability of the membrane were estimated from the rate of osmotic swelling of the cells. In the presence of Cry1C, which is toxic to Sf9 cells, the permeability of the cell membrane to KCl and glucose increased in a dose-dependent manner. In contrast, Cry1Aa, Cry1Ab and Cry1Ac, toxins to which Sf9 cells are not susceptible, had no detectable effect. Pores formed by Cry1C allowed the diffusion of sucrose, but were impermeable to the trisaccharide raffinose. On the basis of the hydrodynamic radii of these substances, the diameter of the pores was estimated to be 1.0-1.2 nm. In the presence of salts, the rate of swelling of cells exposed to Cry1C was about equally influenced by the size of the anion as by that of the cation, indicating that the ionic selectivity of the pores is low.

Cry1F Resistance in Fall Armyworm Spodoptera frugiperda: Single Gene versus Pyramided Bt Maize

PubMed Central

Huang, Fangneng; Qureshi, Jawwad A.; Meagher, Robert L.; Reisig, Dominic D.; Head, Graham P.; Andow, David A.; Ni, Xinzi; Kerns, David; Buntin, G. David; Niu, Ying; Yang, Fei; Dangal, Vikash

2014-01-01

Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. after 18 years of intensive Bt maize planting. Here we report compelling evidence of field resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith), to Cry1F maize (TC 3507) in the southeastern region of the U.S. An F2 screen showed a surprisingly high (0.293) Cry1F resistance allele frequency in a population collected in 2011 from non-Bt maize in south Florida. Field populations from non-Bt maize in 2012–2013 exhibited 18.8-fold to >85.4-fold resistance to purified Cry1F protein and those collected from unexpectedly damaged Bt maize plants at several locations in Florida and North Carolina had >85.4-fold resistance. In addition, reduced efficacy and control failure of Cry1F maize against natural populations of S. frugiperda were documented in field trials using Cry1F-based and pyramided Bt maize products in south Florida. The Cry1F-resistant S. frugiperda also showed a low level of cross-resistance to Cry1A.105 and related maize products, but not to Cry2Ab2 or Vip3A. The occurrence of Cry1F resistance in the U.S. mainland populations of S. frugiperda likely represents migration of insects from Puerto Rico, indicating the great challenges faced in achieving effective resistance management for long-distance migratory pests like S. frugiperda. PMID:25401494

Cry1F resistance in fall armyworm Spodoptera frugiperda: single gene versus pyramided Bt maize.

PubMed

Huang, Fangneng; Qureshi, Jawwad A; Meagher, Robert L; Reisig, Dominic D; Head, Graham P; Andow, David A; Ni, Xinzi; Kerns, David; Buntin, G David; Niu, Ying; Yang, Fei; Dangal, Vikash

2014-01-01

Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. after 18 years of intensive Bt maize planting. Here we report compelling evidence of field resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith), to Cry1F maize (TC 3507) in the southeastern region of the U.S. An F2 screen showed a surprisingly high (0.293) Cry1F resistance allele frequency in a population collected in 2011 from non-Bt maize in south Florida. Field populations from non-Bt maize in 2012-2013 exhibited 18.8-fold to >85.4-fold resistance to purified Cry1F protein and those collected from unexpectedly damaged Bt maize plants at several locations in Florida and North Carolina had >85.4-fold resistance. In addition, reduced efficacy and control failure of Cry1F maize against natural populations of S. frugiperda were documented in field trials using Cry1F-based and pyramided Bt maize products in south Florida. The Cry1F-resistant S. frugiperda also showed a low level of cross-resistance to Cry1A.105 and related maize products, but not to Cry2Ab2 or Vip3A. The occurrence of Cry1F resistance in the U.S. mainland populations of S. frugiperda likely represents migration of insects from Puerto Rico, indicating the great challenges faced in achieving effective resistance management for long-distance migratory pests like S. frugiperda.

Pretreatment with Cry1Ac Protoxin Modulates the Immune Response, and Increases the Survival of Plasmodium-Infected CBA/Ca Mice

PubMed Central

Legorreta-Herrera, Martha; Oviedo Meza, Rodrigo; Moreno-Fierros, Leticia

2010-01-01

Malaria is a major global health problem that kills 1-2 million people each year. Despite exhaustive research, naturally acquired immunity is poorly understood. Cry1A proteins are potent immunogens with adjuvant properties and are able to induce strong cellular and humoral responses. In fact, it has been shown that administration of Cry1Ac protoxin alone or with amoebic lysates induces protection against the lethal infection caused by the protozoa Naegleria fowleri. In this work, we studied whether Cry1Ac is able to activate the innate immune response to induce protection against Plasmodium berghei ANKA (lethal) and P. chabaudi AS (nonlethal) parasites in CBA/Ca mice. Treatment with Cry1Ac induced protection against both Plasmodium species in terms of reduced parasitaemia, longer survival time, modulation of pro- and anti-inflammatory cytokines, and increased levels of specific antibodies against Plasmodium. Understanding how to boost innate immunity to Plasmodium infection should lead to immunologically based intervention strategies. PMID:20300584

Systems-level analysis of risk genes reveals the modular nature of schizophrenia.

PubMed

Liu, Jiewei; Li, Ming; Luo, Xiong-Jian; Su, Bing

2018-05-19

Schizophrenia (SCZ) is a complex mental disorder with high heritability. Genetic studies (especially recent genome-wide association studies) have identified many risk genes for schizophrenia. However, the physical interactions among the proteins encoded by schizophrenia risk genes remain elusive and it is not known whether the identified risk genes converge on common molecular networks or pathways. Here we systematically investigated the network characteristics of schizophrenia risk genes using the high-confidence protein-protein interactions (PPI) from the human interactome. We found that schizophrenia risk genes encode a densely interconnected PPI network (P = 4.15 × 10 -31 ). Compared with the background genes, the schizophrenia risk genes in the interactome have significantly higher degree (P = 5.39 × 10 -11 ), closeness centrality (P = 7.56 × 10 -11 ), betweeness centrality (P = 1.29 × 10 -11 ), clustering coefficient (P = 2.22 × 10 -2 ), and shorter average shortest path length (P = 7.56 × 10 -11 ). Based on the densely interconnected PPI network, we identified 48 hub genes and 4 modules formed by highly interconnected schizophrenia genes. We showed that the proteins encoded by schizophrenia hub genes have significantly more direct physical interactions. Gene ontology (GO) analysis revealed that cell adhesion, cell cycle, immune system response, and GABR-receptor complex categories were enriched in the modules formed by highly interconnected schizophrenia risk genes. Our study reveals that schizophrenia risk genes encode a densely interconnected molecular network and demonstrates the modular nature of schizophrenia. Copyright © 2018 Elsevier B.V. All rights reserved.

Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy

Treesearch

Algimantas P. Valaitis

2011-01-01

The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the...

Bacillus thuringiensis monogenic strains: screening and interactions with insecticides used against rice pests

PubMed Central

Pinto, Laura M.N.; Dörr, Natália C.; Ribeiro, Ana Paula A.; de Salles, Silvia M.; de Oliveira, Jaime V.; Menezes, Valmir G.; Fiuza, Lidia M.

2012-01-01

The screening of Bacillus thuringiensis (Bt) Cry proteins with high potential to control insect pests has been the goal of numerous research groups. In this study, we evaluated six monogenic Bt strains (Bt dendrolimus HD-37, Bt kurstaki HD-1, Bt kurstaki HD-73, Bt thuringiensis 4412, Bt kurstaki NRD-12 and Bt entomocidus 60.5, which codify the cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1C, cry2A genes respectively) as potential insecticides for the most important insect pests of irrigated rice: Spodoptera frugiperda, Diatraea saccharalis, Oryzophagus oryzae, Oebalus poecilus and Tibraca limbativentris. We also analyzed their compatibility with chemical insecticides (thiamethoxam, labdacyhalothrin, malathion and fipronil), which are extensively used in rice crops. The bioassay results showed that Bt thuringiensis 4412 and Bt entomocidus 60.5 were the most toxic for the lepidopterans, with a 93% and 82% mortality rate for S. frugiperda and D. saccharalis, respectively. For O. oryzae, the Bt kurstaki NRD-12 (64%) and Bt dendrolimus HD-37 (62%) strains were the most toxic. The Bt dendrolimus HD-37 strain also caused high mortality (82%) to O. poecilus, however the strains assessed to T. limbativentris caused a maximum rate of 5%. The assays for the Bt strains interaction with insecticides revealed the compatibility of the six strains with the four insecticides tested. The results from this study showed the high potential of cry1Aa and cry1Ba genes for genetic engineering of rice plants or the strains to biopesticide formulations. PMID:24031872

Arabidopsis Casein Kinase1 Proteins CK1.3 and CK1.4 Phosphorylate Cryptochrome2 to Regulate Blue Light Signaling[C][W

PubMed Central

Tan, Shu-Tang; Dai, Cheng; Liu, Hong-Tao; Xue, Hong-Wei

2013-01-01

Casein kinase1 (CK1) plays crucial roles in regulating growth and development via phosphorylating various substrates throughout the eukaryote kingdom. Blue light is crucial for normal growth of both plants and animals, and blue light receptor cryptochrome2 (CRY2) undergoes blue light–dependent phosphorylation and degradation in planta. To study the function of plant CK1s, systematic genetic analysis showed that deficiency of two paralogous Arabidopsis thaliana CK1s, CK1.3 and CK1.4, caused shortened hypocotyls, especially under blue light, while overexpression of either CK1.3 or CK1.4 resulted in the insensitive response to blue light and delayed flowering under long-day conditions. CK1.3 or CK1.4 act dependently on CRY2, and overexpression of CK1.3 or CK1.4 significantly suppresses the hypersensitive response to blue light by CRY2 overexpression. Biochemical studies showed that CK1.3 and CK1.4 directly phosphorylate CRY2 at Ser-587 and Thr-603 in vitro and negatively regulate CRY2 stability in planta, which are stimulated by blue light, further confirming the crucial roles of CK1.3 and CK1.4 in blue light responses through phosphorylating CRY2. Interestingly, expression of CK1.3 and CK1.4 is stimulated by blue light and feedback regulated by CRY2-mediated signaling. These results provide direct evidence for CRY2 phosphorylation and informative clues on the mechanisms of CRY2-mediated light responses. PMID:23897926

Comparative performance of modified full-length and truncated Bacillus thuringiensis-cry1Ac genes in transgenic tomato.

PubMed

Koul, Bhupendra; Yadav, Reena; Sanyal, Indraneel; Amla, Devindra Vijay

2015-01-01

Bt-cry1Ac gene has been reputedly effective against Helicoverpa armigera a notorious lepidopteran pest. Reports on the expression of full-length and truncated cry1Ac genes in plants for effective resistance against Helicoverpa sp. have been documented however, their performance is still ambiguous. Moreover, the question remains to be addressed that truncation of 3' end of the native gene was documented and suggested for active insecticidal toxin production while the most successful transgenic event(s) of commercialized-cotton are based on full-length of the cry gene. Therefore, we performed a comparative study on the efficacy of the two versions of cry1Ac genes (full-length: 3,510 bp and truncated: 1,845 bp) in T0 and T1 transgenic tomato plants and analyzed the extent of protection against H. armigera and also compared the results with our previous findings related to a successful transgenic tomato line Ab25E, expressing cry1Ab gene. The integration of cry1Ac gene(s) in T0 transgenic plants and its inheritance in T1 progeny was observed by PCR, RT-PCR and Southern blot hybridization analysis while, the toxin integrity, expression and toxicity was monitored by Western immunoassay, DAS-ELISA and insect bioassay respectively. An average transformation frequency and Bt-Cry protein content of 16.93 ± 2.10 and 0.0020-0.0128% of total soluble protein (TSP) was obtained with pRD400 vector (Trcry1Ac) while, a much lower value of 9.30 ± 2.041 and 0.0001 - 0.0026% of TSP was observed with pNBRI-1 vector (Flcry1Ac), respectively. The promising Trcry1Ac T0 transgenic plants and their T1 progeny gave full protection from H. armigera. Although Flcry1Ac gene showed lower transformation frequency and lower expression, it showed higher toxicity to H. armigera when compared with truncated Trcry1Ac gene. The full-length cry1Ac gene can be redesigned for higher expression and performance in dicots or a hybrid gene could be designed having a blend of strong receptor binding and stable expression characteristics for enhanced efficacy and toxicity to the susceptible insects.

Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis).

PubMed

de Oliveira, Raquel S; Oliveira-Neto, Osmundo B; Moura, Hudson F N; de Macedo, Leonardo L P; Arraes, Fabrício B M; Lucena, Wagner A; Lourenço-Tessutti, Isabela T; de Deus Barbosa, Aulus A; da Silva, Maria C M; Grossi-de-Sa, Maria F

2016-01-01

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests.

Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis)

PubMed Central

de Oliveira, Raquel S.; Oliveira-Neto, Osmundo B.; Moura, Hudson F. N.; de Macedo, Leonardo L. P.; Arraes, Fabrício B. M.; Lucena, Wagner A.; Lourenço-Tessutti, Isabela T.; de Deus Barbosa, Aulus A.; da Silva, Maria C. M.; Grossi-de-Sa, Maria F.

2016-01-01

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests. PMID:26925081

An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coli SAB11

PubMed Central

Bearden, Scott W.; Staggs, Teanna M.; Perry, Robert D.

1998-01-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media. PMID:9495751

An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli SAB11.

PubMed

Bearden, S W; Staggs, T M; Perry, R D

1998-03-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The approximately 21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

Molecular characterization of Bacillus thuringiensis isolated from diverse habitats of India.

PubMed

Patel, Ketan D; Chudasama, Chaitanyasinh J; Ingle, Sanjay S

2012-08-01

Bacillus thuringiensis (Bt) strains were isolated from 94 samples from different geographical regions. Novel types of crystalline inclusion bodies were observed from some of the isolates. Crystalline inclusions of bipyramidal, spherical and cuboidal morphology were found produced by most of the isolates. Isolate GS12 showed crystal on one side of spore while isolate GM108 formed crystals on both termini of spore. Isolate GN31 produced large sized bipyramidal crystals. SDS-PAGE analysis of the spore crystal suspension showed major protein bands in the range of 29 and 140 kDa. Two new serovars of Bt viz. GS4 and GN24 having H3abce and H3ab serotype respectively were isolated. Toxicity comparable to the reference strain Bacillus thuringiensis subs. kurstaki (Btk) HD-1 was observed for the isolates GM20, GM17 and MP3 against larvae of Helicoverpa armigera. Some of the isolates harboring cry genes like cry1Ac and cry2 did not show any toxicity towards H. armigera while most of the isolates were harboring cry1, cry1Ac and cry2 gene. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acquisition of Cry1Ac Protein by Non-Target Arthropods in Bt Soybean Fields

PubMed Central

Yu, Huilin; Romeis, Jörg; Li, Yunhe; Li, Xiangju; Wu, Kongming

2014-01-01

Soybean tissue and arthropods were collected in Bt soybean fields in China at different times during the growing season to investigate the exposure of arthropods to the plant-produced Cry1Ac toxin and the transmission of the toxin within the food web. Samples from 52 arthropod species/taxa belonging to 42 families in 10 orders were analysed for their Cry1Ac content using enzyme-linked immunosorbent assay (ELISA). Among the 22 species/taxa for which three samples were analysed, toxin concentration was highest in the grasshopper Atractomorpha sinensis and represented about 50% of the concentration in soybean leaves. Other species/taxa did not contain detectable toxin or contained a concentration that was between 1 and 10% of that detected in leaves. These Cry1Ac-positive arthropods included a number of mesophyll-feeding Hemiptera, a cicadellid, a curculionid beetle and, among the predators, a thomisid spider and an unidentified predatory bug belonging to the Anthocoridae. Within an arthropod species/taxon, the Cry1Ac content sometimes varied between life stages (nymphs/larvae vs. adults) and sampling dates (before, during, and after flowering). Our study is the first to provide information on Cry1Ac-expression levels in soybean plants and Cry1Ac concentrations in non-target arthropods in Chinese soybean fields. The data will be useful for assessing the risk of non-target arthropod exposure to Cry1Ac in soybean. PMID:25110881

IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwasnicka-Crawford, Dorota A.; Carson, Andrew R.; Scherer, Stephen W.

The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNAmore » is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions.« less

Ingestion of Bt rice pollen does not reduce the survival or hypopharyngeal gland development of Apis mellifera adults.

PubMed

Wang, Yuanyuan; Dai, Pingli; Chen, Xiuping; Romeis, Jörg; Shi, Jianrong; Peng, Yufa; Li, Yunhe

2017-05-01

Because of its ecological and economic importance, the honey bee Apis mellifera is commonly used to assess the environmental risk of insect-resistant, genetically modified plants. In the present study, feeding-exposure experiments were used to determine whether pollen from transgenic rice harms A. mellifera worker bees. In 1 experiment, the survival and mean acinus diameter of hypopharyngeal glands of adult bees were similar when bees were fed on pollen from Bt rice lines or from a non-Bt rice line, but bee survival was significantly reduced when they received pollen that was mixed with potassium arsenate as a positive control. In a second experiment, bee survival and hypopharyngeal gland development were not reduced when adult bees were fed on non-Bt pollen and a sucrose solution supplemented with Cry2A at 400 µg/g, Cry1C at 50 µg/g, or bovine serum albumin (BSA) at 400 µg/g, but bee survival and hypopharyngeal gland development were reduced when the diet was supplemented with soybean trypsin inhibitor as a positive control. In both experiments, the uptake of Cry proteins by adult bees was confirmed. Overall, the results indicate that the planting of Bt rice lines expressing Cry2A or Cry1C protein poses a negligible risk to A. mellifera worker bees. Environ Toxicol Chem 2017;36:1243-1248. © 2016 SETAC. © 2016 SETAC.

Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein

PubMed Central

Poppers, Jeremy; Mulvey, Matthew; Khoo, David; Mohr, Ian

2000-01-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation. PMID:11070019

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

PubMed

Poppers, J; Mulvey, M; Khoo, D; Mohr, I

2000-12-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

Comparative analysis of Bacillus thuringiensis toxin binding to gypsy moth, browntail moth, and douglas-fir tussock moth midgut tissue sections using fluorescence microscopy

Treesearch

Algimantas P. Valaitis; John D. Podgwaite

2011-01-01

Many strains of Bacillus thuringiensis (Bt) produce insecticidal proteins, also referred to as Cry toxins, in crystal inclusions during sporulation. When ingested by insects, the Cry toxins bind to receptors on the brush border midgut epithelial cells and create pores in the epithelial gut membranes resulting in the death of...

Assessing the potential for interaction between the insecticidal activity of two genetically engineered cotton events combined by conventional breeding: An example with COT102 × MON 15985.

PubMed

Levine, Steven L; Mueller, Geoffrey M; Uffman, Joshua P

2016-08-01

Bollgard(®) III was developed by combining cotton events COT102 and MON 15985 through conventional breeding to improve efficacy against lepidopteran feeding damage. COT102 produces the Vip3Aa19 protein and MON 15985 produces the Cry1Ac and Cry2Ab2 proteins. COT102 × MON 15985 has also been bred with Roundup Ready Flex(®) cotton (MON 88913) that confers glyphosate tolerance. This study evaluated the activity of COT102 and MON 15985 and the combined activity of COT102 and MON 15985 against the cotton bollworm (CBW, Helicoverpa zea). COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 have comparable Vip3Aa19 and/or Cry1Ac, Cry2Ab2 protein expression levels as determined by enzyme-linked immunosorbent assay. CBW demonstrated concentration-dependent growth inhibition after 7-days of feeding on lyophilized leaf tissue derived from COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 incorporated into an artificial diet. Observed EC50 values for COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 were comparable (≤4% deviation) with the predicted EC50 value under the assumption of additivity using the combined activity of COT102 and MON 15985. No interaction in biological activity between COT102 and MON 15985 is consistent with results from competition and ligand blotting assays that demonstrated that Vip3Aa does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. The results from this study demonstrate that the activity of COT102 × MON 15985 against CBW is consistent with predictions of additivity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Neurobiology of culturally common maternal responses to infant cry

PubMed Central

Bornstein, Marc H.; Rigo, Paola; Esposito, Gianluca; Swain, James E.; Suwalsky, Joan T. D.; Su, Xueyun; Du, Xiaoxia; Zhang, Kaihua; Cote, Linda R.; De Pisapia, Nicola; Venuti, Paola

2017-01-01

This report coordinates assessments of five types of behavioral responses in new mothers to their own infants’ cries with neurobiological responses in new mothers to their own infants’ cries and in experienced mothers and inexperienced nonmothers to infant cries and other emotional and control sounds. We found that 684 new primipara mothers in 11 countries (Argentina, Belgium, Brazil, Cameroon, France, Kenya, Israel, Italy, Japan, South Korea, and the United States) preferentially responded to their infants’ vocalizing distress by picking up and holding and by talking to their infants, as opposed to displaying affection, distracting, or nurturing. Complementary functional magnetic resonance imaging (fMRI) analyses of brain responses to their own infants’ cries in 43 new primipara US mothers revealed enhanced activity in concordant brain territories linked to the intention to move and to speak, to process auditory stimulation, and to caregive [supplementary motor area (SMA), inferior frontal regions, superior temporal regions, midbrain, and striatum]. Further, fMRI brain responses to infant cries in 50 Chinese and Italian mothers replicated, extended, and, through parcellation, refined the results. Brains of inexperienced nonmothers activated differently. Culturally common responses to own infant cry coupled with corresponding fMRI findings to own infant and to generic infant cries identified specific, common, and automatic caregiving reactions in mothers to infant vocal expressions of distress and point to their putative neurobiological bases. Candidate behaviors embedded in the nervous systems of human caregivers lie at the intersection of evolutionary biology and developmental cultural psychology. PMID:29078366

Emergency Department Triage of the "Incessantly Crying" Baby.

PubMed

Chua, Caroline; Setlik, Jennifer; Niklas, Victoria

2016-11-01

Incessant crying is one of the most common caregiver complaints during emergency department (ED) visits in the first few months of the child's life. Although the majority of cases are attributed to normal infant behavior, the differential diagnosis remains broad. Moreover, the potential for the negative impact of incessant crying on the mental well-being of caregivers as well as the infants necessitates that complaints be taken seriously and that "red flags" for underlying organic causes be ruled out and caregiver anxiety quelled. In addition, the apparent triviality of incessant crying in the face of the life-threatening illnesses or injuries that confront practitioners in the ED necessitates a high level of due diligence in the evaluation of these infants and their families. Ensuring the availability of family support is essential in the discharge planning. Families should also perceive the empathy of the physician and feel reassured about their safe discharge home. Although it is a challenge to examine an incessantly crying infant in all care settings, the failure to recognize the small percentage of infants that present with incessant crying as a manifestation of an underlying organic illness may have grave consequences. [Pediatr Ann. 2016;45(11):e394-e398.]. Copyright 2016, SLACK Incorporated.

Estimating Foodborne Gastroenteritis, Australia

PubMed Central

Kirk, Martyn D.; Becker, Niels; Gregory, Joy E.; Unicomb, Leanne; Millard, Geoffrey; Stafford, Russell; Lalor, Karin

2005-01-01

We estimated for Australia the number of cases, hospitalizations, and deaths due to foodborne gastroenteritis in a typical year, circa 2000. The total amount of infectious gastroenteritis was measured by using a national telephone survey. The foodborne proportion was estimated from Australian data on each of 16 pathogens. To account for uncertainty, we used simulation techniques to calculate 95% credibility intervals (CrI). The estimate of incidence of gastroenteritis in Australia is 17.2 million (95% confidence interval 14.5–19.9 million) cases per year. We estimate that 32% (95% CrI 24%–40%) are foodborne, which equals 0.3 (95% CrI 0.2–0.4) episodes per person, or 5.4 million (95% CrI 4.0–6.9 million) cases annually in Australia. Norovirus, enteropathogenic Escherichia coli, Campylobacter spp., and Salmonella spp. cause the most illnesses. In addition, foodborne gastroenteritis causes ≈15,000 (95% CrI 11,000–18,000) hospitalizations and 80 (95% CrI 40–120) deaths annually. This study highlights global public health concerns about foodborne diseases and the need for standardized methods, including assessment of uncertainty, for international comparison. PMID:16102316

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

PubMed

Sidorenko, Lyudmila V; Lee, Tzuu-Fen; Woosley, Aaron; Moskal, William A; Bevan, Scott A; Merlo, P Ann Owens; Walsh, Terence A; Wang, Xiujuan; Weaver, Staci; Glancy, Todd P; Wang, PoHao; Yang, Xiaozeng; Sriram, Shreedharan; Meyers, Blake C

2017-11-01

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Effects of a diet containing genetically modified rice expressing the Cry1Ab/1Ac protein (Bacillus thuringiensis toxin) on broiler chickens.

PubMed

Li, Zeyang; Gao, Yang; Zhang, Minhong; Feng, Jinghai; Xiong, Yandan

2015-01-01

The aim of this study was to evaluate the effect of feeding Bacillus thuringiensis (Bt) rice expressing the Cry1Ab/1Ac protein on broiler chicken. The genetically modified (GM) Bt rice was compared with the corresponding non-GM rice regarding performance of feeding groups, their health status, relative organ weights, biochemical serum parameters and occurrence of Cry1Ab/1Ac gene fragments. One hundred and eighty day-old Arbor Acres female broilers with the same health condition were randomly allocated to the two treatments (6 replicate cages with 15 broilers in each cage per treatment). They received diets containing GM rice (GM group) or its parental non-GM rice (non-GM group) at 52-57% of the air-dried diet for 42 days. The results show that the transgenic rice had a similar nutrient composition as the non-GM rice and had no adverse effects on chicken growth, biochemical serum parameters and necropsy during the 42-day feeding period. In birds fed the GM rice, no transgenic gene fragments were detected in the samples of blood, liver, kidneys, spleen, jejunum, ileum, duodenum and muscle tissue. In conclusion, the results suggest that Bt rice expressing Cry1Ab/1Ac protein has no adverse effects on broiler chicken. Therefore, it can be considered as safe and used as feed source for broiler chicken.

Lack of Detectable Allergenicity in Genetically Modified Maize Containing “Cry” Proteins as Compared to Native Maize Based on In Silico & In Vitro Analysis

PubMed Central

Mathur, Chandni; Kathuria, Pooran C.; Dahiya, Pushpa; Singh, Anand B.

2015-01-01

Background Genetically modified, (GM) crops with potential allergens must be evaluated for safety and endogenous IgE binding pattern compared to native variety, prior to market release. Objective To compare endogenous IgE binding proteins of three GM maize seeds containing Cry 1Ab,1Ac,1C transgenic proteins with non GM maize. Methods An integrated approach of in silico & in vitro methods was employed. Cry proteins were tested for presence of allergen sequence by FASTA in allergen databases. Biochemical assays for maize extracts were performed. Specific IgE (sIgE) and Immunoblot using food sensitized patients sera (n = 39) to non GM and GM maize antigens was performed. Results In silico approaches, confirmed for non sequence similarity of stated transgenic proteins in allergen databases. An insignificant (p> 0.05) variation in protein content between GM and non GM maize was observed. Simulated Gastric Fluid (SGF) revealed reduced number of stable protein fractions in GM then non GM maize which might be due to shift of constituent protein expression. Specific IgE values from patients showed insignificant difference in non GM and GM maize extracts. Five maize sensitized cases, recognized same 7 protein fractions of 88-28 kD as IgE bindng in both GM and non-GM maize, signifying absence of variation. Four of the reported IgE binding proteins were also found to be stable by SGF. Conclusion Cry proteins did not indicate any significant similarity of >35% in allergen databases. Immunoassays also did not identify appreciable differences in endogenous IgE binding in GM and non GM maize. PMID:25706412

A role for cryptochromes in sleep regulation.

PubMed

Wisor, Jonathan P; O'Hara, Bruce F; Terao, Akira; Selby, Chris P; Kilduff, Thomas S; Sancar, Aziz; Edgar, Dale M; Franken, Paul

2002-12-20

The cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the generation of circadian rhythms, as mice lacking both of these genes (cry1,2-/-) lack circadian rhythms. We studied sleep in cry1,2-/- mice under baseline conditions as well as under conditions of constant darkness and enforced wakefulness to determine whether cryptochromes influence sleep regulatory processes. Under all three conditions, cry1,2-/- mice exhibit the hallmarks of high non-REM sleep (NREMS) drive (i.e., increases in NREMS time, NREMS consolidation, and EEG delta power during NREMS). This unexpected phenotype was associated with elevated brain mRNA levels of period 1 and 2 (per1,2), and albumin d-binding protein (dbp), which are known to be transcriptionally inhibited by CRY1,2. To further examine the relationship between circadian genes and sleep homeostasis, we examined wild type mice and rats following sleep deprivation and found increased levels of per1,2 mRNA and decreased levels of dbp mRNA specifically in the cerebral cortex; these changes subsided with recovery sleep. The expression of per3, cry1,2, clock, npas2, bmal1, and casein-kinase-1epsilon did not change with sleep deprivation. These results indicate that mice lacking cryptochromes are not simply a genetic model of circadian arrhythmicity in rodents and functionally implicate cryptochromes in the homeostatic regulation of sleep.

Decomposition rates and residue-colonizing microbial communities of Bacillus thuringiensis insecticidal protein Cry3Bb-expressing (Bt) and non-Bt corn hybrids in the field.

PubMed

Xue, Kai; Serohijos, Raquel C; Devare, Medha; Thies, Janice E

2011-02-01

Despite the rapid adoption of crops expressing the insecticidal Cry protein(s) from Bacillus thuringiensis (Bt), public concern continues to mount over the potential environmental impacts. Reduced residue decomposition rates and increased tissue lignin concentrations reported for some Bt corn hybrids have been highlighted recently as they may influence soil carbon dynamics. We assessed the effects of MON863 Bt corn, producing the Cry3Bb protein against the corn rootworm complex, on these aspects and associated decomposer communities by terminal restriction fragment length polymorphism (T-RFLP) analysis. Litterbags containing cobs, roots, or stalks plus leaves from Bt and unmodified corn with (non-Bt+I) or without (non-Bt) insecticide applied were placed on the soil surface and at a 10-cm depth in field plots planted with these crop treatments. The litterbags were recovered and analyzed after 3.5, 15.5, and 25 months. No significant effect of treatment (Bt, non-Bt, and non-Bt+I) was observed on initial tissue lignin concentrations, litter decomposition rate, or bacterial decomposer communities. The effect of treatment on fungal decomposer communities was minor, with only 1 of 16 comparisons yielding separation by treatment. Environmental factors (litterbag recovery year, litterbag placement, and plot history) led to significant differences for most measured variables. Combined, these results indicate that the differences detected were driven primarily by environmental factors rather than by any differences between the corn hybrids or the use of tefluthrin. We conclude that the Cry3Bb corn tested in this study is unlikely to affect carbon residence time or turnover in soils receiving these crop residues.

Effects of Transgenic Cry1Ac + CpTI Cotton on Non-Target Mealybug Pest Ferrisia virgata and Its Predator Cryptolaemus montrouzieri

PubMed Central

Wu, Hongsheng; Zhang, Yuhong; Liu, Ping; Xie, Jiaqin; He, Yunyu; Deng, Congshuang; De Clercq, Patrick; Pang, Hong

2014-01-01

Recently, several invasive mealybugs (Hemiptera: Pseudococcidae) have rapidly spread to Asia and have become a serious threat to the production of cotton including transgenic cotton. Thus far, studies have mainly focused on the effects of mealybugs on non-transgenic cotton, without fully considering their effects on transgenic cotton and trophic interactions. Therefore, investigating the potential effects of mealybugs on transgenic cotton and their key natural enemies is vitally important. A first study on the effects of transgenic cotton on a non-target mealybug, Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae) was performed by comparing its development, survival and body weight on transgenic cotton leaves expressing Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) with those on its near-isogenic non-transgenic line. Furthermore, the development, survival, body weight, fecundity, adult longevity and feeding preference of the mealybug predator Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) was assessed when fed F. virgata maintained on transgenic cotton. In order to investigate potential transfer of Cry1Ac and CpTI proteins via the food chain, protein levels in cotton leaves, mealybugs and ladybirds were quantified. Experimental results showed that F. virgata could infest this bivalent transgenic cotton. No significant differences were observed in the physiological parameters of the predator C. montrouzieri offered F. virgata reared on transgenic cotton or its near-isogenic line. Cry1Ac and CpTI proteins were detected in transgenic cotton leaves, but no detectable levels of both proteins were present in the mealybug or its predator when reared on transgenic cotton leaves. Our bioassays indicated that transgenic cotton poses a negligible risk to the predatory coccinellid C. montrouzieri via its prey, the mealybug F. virgata. PMID:24751821

Effects of transgenic Cry1Ac + CpTI cotton on non-target mealybug pest Ferrisia virgata and its predator Cryptolaemus montrouzieri.

PubMed

Wu, Hongsheng; Zhang, Yuhong; Liu, Ping; Xie, Jiaqin; He, Yunyu; Deng, Congshuang; De Clercq, Patrick; Pang, Hong

2014-01-01

Recently, several invasive mealybugs (Hemiptera: Pseudococcidae) have rapidly spread to Asia and have become a serious threat to the production of cotton including transgenic cotton. Thus far, studies have mainly focused on the effects of mealybugs on non-transgenic cotton, without fully considering their effects on transgenic cotton and trophic interactions. Therefore, investigating the potential effects of mealybugs on transgenic cotton and their key natural enemies is vitally important. A first study on the effects of transgenic cotton on a non-target mealybug, Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae) was performed by comparing its development, survival and body weight on transgenic cotton leaves expressing Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) with those on its near-isogenic non-transgenic line. Furthermore, the development, survival, body weight, fecundity, adult longevity and feeding preference of the mealybug predator Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) was assessed when fed F. virgata maintained on transgenic cotton. In order to investigate potential transfer of Cry1Ac and CpTI proteins via the food chain, protein levels in cotton leaves, mealybugs and ladybirds were quantified. Experimental results showed that F. virgata could infest this bivalent transgenic cotton. No significant differences were observed in the physiological parameters of the predator C. montrouzieri offered F. virgata reared on transgenic cotton or its near-isogenic line. Cry1Ac and CpTI proteins were detected in transgenic cotton leaves, but no detectable levels of both proteins were present in the mealybug or its predator when reared on transgenic cotton leaves. Our bioassays indicated that transgenic cotton poses a negligible risk to the predatory coccinellid C. montrouzieri via its prey, the mealybug F. virgata.

Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy

PubMed Central

McLendon, Patrick M.; Ferguson, Bradley S.; Osinska, Hanna; Bhuiyan, Md. Shenuarin; James, Jeanne; McKinsey, Timothy A.; Robbins, Jeffrey

2014-01-01

Proteinopathy causes cardiac disease, remodeling, and heart failure but the pathological mechanisms remain obscure. Mutated αB-crystallin (CryABR120G), when expressed only in cardiomyocytes in transgenic (TG) mice, causes desmin-related cardiomyopathy, a protein conformational disorder. The disease is characterized by the accumulation of toxic misfolded protein species that present as perinuclear aggregates known as aggresomes. Previously, we have used the CryABR120G model to determine the underlying processes that result in these pathologic accumulations and to explore potential therapeutic windows that might be used to decrease proteotoxicity. We noted that total ventricular protein is hypoacetylated while hyperacetylation of α-tubulin, a substrate of histone deacetylase 6 (HDAC6) occurs. HDAC6 has critical roles in protein trafficking and autophagy, but its function in the heart is obscure. Here, we test the hypothesis that tubulin acetylation is an adaptive process in cardiomyocytes. By modulating HDAC6 levels and/or activity genetically and pharmacologically, we determined the effects of tubulin acetylation on aggregate formation in CryABR120G cardiomyocytes. Increasing HDAC6 accelerated aggregate formation, whereas siRNA-mediated knockdown or pharmacological inhibition ameliorated the process. HDAC inhibition in vivo induced tubulin hyperacetylation in CryABR120G TG hearts, which prevented aggregate formation and significantly improved cardiac function. HDAC6 inhibition also increased autophagic flux in cardiomyocytes, and increased autophagy in the diseased heart correlated with increased tubulin acetylation, suggesting that autophagy induction might underlie the observed cardioprotection. Taken together, our data suggest a mechanistic link between tubulin hyperacetylation and autophagy induction and points to HDAC6 as a viable therapeutic target in cardiovascular disease. PMID:25404307

In Vivo and In Vitro Binding of Vip3Aa to Spodoptera frugiperda Midgut and Characterization of Binding Sites by 125I Radiolabeling

PubMed Central

Chakroun, Maissa

2014-01-01

Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the binding protocol for the 125I-Vip3Aa to S. frugiperda brush border membrane vesicles (BBMV) allowed the determination of binding parameters of Vip3A proteins for the first time. Heterologous competition using Vip3Ad, Vip3Ae, and Vip3Af as competitor proteins showed that they share the same binding site with Vip3Aa. In contrast, when using Cry1Ab and Cry1Ac as competitors, no competitive binding was observed, which makes them appropriate candidates to be used in combination with Vip3A proteins in transgenic crops. PMID:25002420

Extrapolating non-target risk of Bt crops from laboratory to field.

PubMed

Duan, Jian J; Lundgren, Jonathan G; Naranjo, Steve; Marvier, Michelle

2010-02-23

The tiered approach to assessing ecological risk of insect-resistant transgenic crops assumes that lower tier laboratory studies, which expose surrogate non-target organisms to high doses of insecticidal proteins, can detect harmful effects that might be manifested in the field. To test this assumption, we performed meta-analyses comparing results for non-target invertebrates exposed to Bacillus thuringiensis (Bt) Cry proteins in laboratory studies with results derived from independent field studies examining effects on the abundance of non-target invertebrates. For Lepidopteran-active Cry proteins, laboratory studies correctly predicted the reduced field abundance of non-target Lepidoptera. However, laboratory studies incorporating tri-trophic interactions of Bt plants, herbivores and parasitoids were better correlated with the decreased field abundance of parasitoids than were direct-exposure assays. For predators, laboratory tri-trophic studies predicted reduced abundances that were not realized in field studies and thus overestimated ecological risk. Exposure to Coleopteran-active Cry proteins did not significantly reduce the laboratory survival or field abundance of any functional group examined. Our findings support the assumption that laboratory studies of transgenic insecticidal crops show effects that are either consistent with, or more conservative than, those found in field studies, with the important caveat that laboratory studies should explore all ecologically relevant routes of exposure.

Recent advancement on chemical arsenal of Bt toxin and its application in pest management system in agricultural field.

PubMed

Chattopadhyay, Pritam; Banerjee, Goutam

2018-04-01

Bacillus thuringiensis ( Bt ) is a Gram-positive, spore-forming, soil bacterium, which is very popular bio-control agent in agricultural and forestry. In general, B. thuringiensis secretes an array of insecticidal proteins including toxins produced during vegetative growth phase (such as secreted insecticidal protein, Sip; vegetative insecticidal proteins, Vip), parasporal crystalline δ-endotoxins produced during vegetative stationary phase (such as cytolytic toxin, Cyt; and crystal toxin, Cry), and β-exotoxins. Till date, a wide spectrum of Cry proteins has been reported and most of them belong to three-domain-Cry toxins, Bin-like toxin, and Etx_Mtx2-like toxins. To the best of our knowledge, neither Bt insecticidal toxins are exclusive to Bt nor all the strains of Bt are capable of producing insecticidal Bt toxins. The lacuna in their latest classification has also been discussed. In this review, the updated information regarding the insecticidal Bt toxins and their different mode of actions were summarized. Before applying the Bt toxins on agricultural field, the non-specific effects of toxins should be investigated. We also have summarized the problem of insect resistance and the strategies to combat with this problem. We strongly believe that this information will help a lot to the budding researchers in the field of modern pest control biotechnology.

Post-Transcriptional Regulation of the Trypanosome Heat Shock Response by a Zinc Finger Protein

PubMed Central

Droll, Dorothea; Minia, Igor; Fadda, Abeer; Singh, Aditi; Stewart, Mhairi; Queiroz, Rafael; Clayton, Christine

2013-01-01

In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3′-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures. PMID:23592996

Catalytic diversity and homotropic allostery of two Cytochrome P450 monooxygenase like proteins from Trichoderma brevicompactum.

PubMed

Hussain, Razak; Kumari, Indu; Sharma, Shikha; Ahmed, Mushtaq; Khan, Tabreiz Ahmad; Akhter, Yusuf

2017-12-01

Trichothecenes are the secondary metabolites produced by Trichoderma spp. Some of these molecules have been reported for their ability to stimulate plant growth by suppressing plant diseases and hence enabling Trichoderma spp. to be efficiently used as biocontrol agents in modern agriculture. Many of the proteins involved in the trichothecenes biosynthetic pathway in Trichoderma spp. are encoded by the genes present in the tri cluster. Tri4 protein catalyzes three consecutive oxygenation reaction steps during biosynthesis of isotrichodiol in the trichothecenes biosynthetic pathway, while tri11 protein catalyzes the C4 hydroxylation of 12, 13-epoxytrichothec-9-ene to produce trichodermol. In the present study, we have homology modelled the three-dimensional structures of tri4 and tri11 proteins. Furthermore, molecular dynamics simulations were carried out to elucidate the mechanism of their action. Both tri4 and tri11 encode for cytochrome P450 monooxygenase like proteins. These data also revealed effector-induced allosteric changes on substrate binding at an alternative binding site and showed potential homotropic negative cooperativity. These analyses also showed that their catalytic mechanism relies on protein-ligand and protein-heme interactions controlled by hydrophobic and hydrogen-bonding interactions which orient the complex in optimal conformation within the active sites.

RNA Binding Protein RBM38 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19, Which Facilitates Viral DNA Replication.

PubMed

Ganaie, Safder S; Chen, Aaron Yun; Huang, Chun; Xu, Peng; Kleiboeker, Steve; Du, Aifang; Qiu, Jianming

2018-04-15

Human parvovirus B19 (B19V) expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternative polyadenylation to generate 12 viral mRNA transcripts that encode two structural proteins (VP1 and VP2) and three nonstructural proteins (NS1, 7.5-kDa protein, and 11-kDa protein). Splicing at the second 5' donor site (D2 site) of the B19V pre-mRNA is essential for the expression of VP2 and the 11-kDa protein. We previously identified that cis -acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates the recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for the expression of the 11-kDa viral nonstructural protein. We found that ISE2 harbors a consensus RNA binding motif protein 38 (RBM38) binding sequence, 5'-UGUGUG-3'. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that RBM38 binds specifically with the ISE2 element in vitro The knockdown of RBM38 significantly decreases the level of spliced mRNA at D2 that encodes the 11-kDa protein but not that of the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, the knockdown of RBM38 decreases virus replication via downregulating 11-kDa protein expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication and that RBM38 is an essential host factor for B19V pre-mRNA splicing and for the expression of the 11-kDa protein. IMPORTANCE B19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immunocompromised patients and sickle cell disease patients, myocarditis, and hydrops fetalis in pregnant women. Human erythroid progenitor cells (EPCs) are most susceptible to B19V infection and fully support viral DNA replication. The exclusive tropism of B19V for erythroid-lineage cells is dependent not only on the expression of viral receptors and coreceptors on the cell surface but also on the intracellular host factors that support B19V replication. Our present study shows that B19V uses a host factor, RNA binding motif protein 38 (RBM38), for the processing of its pre-mRNA during virus replication. Specifically, RBM38 interacts with the intronic splicing enhancer 2 (ISE2) element of B19V pre-mRNA and promotes 11-kDa protein expression, thereby regulating the 11-kDa protein-mediated augmentation of B19V replication. The identification of this novel host-pathogen interaction will provide mechanistic insights into B19V replication and aid in finding new targets for anti-B19V therapeutics. Copyright © 2018 American Society for Microbiology.

Compatibility of garlic (Allium sativum L.) leaf agglutinin and Cry1Ac δ-endotoxin for gene pyramiding.

PubMed

Upadhyay, Santosh Kumar; Singh, Seema; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna Kumar

2012-03-01

δ-Endotoxins produced by Bacillus thuringiensis (Bt) have been used as bio-pesticides for the control of lepidopteran insect pests. Garlic (Allium sativum L.) leaf agglutinin (ASAL), being toxic to several sap-sucking pests and some lepidopteran pests, may be a good candidate for pyramiding with δ-endotoxins in transgenic plants for enhancing the range of resistance to insect pests. Since ASAL shares the midgut receptors with Cry1Ac in Helicoverpa armigera, there is possibility of antagonism in their toxicity. Our study demonstrated that ASAL increased the toxicity of Cry1Ac against H. armigera while Cry1Ac did not alter the toxicity of ASAL against cotton aphids. The two toxins interacted and increased binding of each other to brush border membrane vesicle (BBMV) proteins and to the two important receptors, alkaline phosphatase (ALP) and aminopeptidase N (APN). The results indicated that the toxins had different binding sites on the ALP and APN but influenced mutual binding. We conclude that ASAL can be safely employed with Cry1Ac for developing transgenic crops for wider insect resistance.

Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin.

PubMed

Tanaka, Shiho; Endo, Haruka; Adegawa, Satomi; Iizuka, Ami; Imamura, Kazuhiro; Kikuta, Shingo; Sato, Ryoichi

2017-12-01

Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that 770 DYWL 773 of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around 770 DYWL 773 of ECL 4 in the ABCC2. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cry1Ab-expressing rice did not influence expression of fecundity-related genes in the wolf spider Pardosa pseudoannulata.

PubMed

Wang, Juan; Peng, Yuan-De; He, Chao; Wei, Bao-Yang; Liang, Yun-Shan; Yang, Hui-Lin; Wang, Zhi; Stanley, David; Song, Qi-Sheng

2016-10-30

The impact of Bacillus thuringiensis (Bt) toxin proteins on non-target predatory arthropods is not well understood at the cellular and molecular levels. Here, we investigated the potential effects of Cry1Ab expressing rice on fecundity of the wolf spider, Pardosa pseudoannulata, and some of the underlying molecular mechanisms. The results indicated that brown planthoppers (BPHs) reared on Cry1Ab-expressing rice accumulated the Cry toxin and that reproductive parameters (pre-oviposition period, post-oviposition stage, number of eggs, and egg hatching rate) of the spiders that consumed BPHs reared on Bt rice were not different from those that consumed BPHs reared on the non-Bt control rice. The accumulated Cry1Ab did not influence several vitellin (Vt) parameters, including stored energy and amino acid composition, during one generation. We considered the possibility that the Cry toxins exert their influence on beneficial predators via more subtle effects detectable at the molecular level in terms of gene expression. This led us to transcriptome analysis to detect differentially expressed genes in the ovaries of spiders exposed to dietary Cry1Ab and their counterpart control spiders. Eight genes, associated with vitellogenesis, vitellogenin receptor activity, and vitellin membrane formation were not differentially expressed between ovaries from the treated and control spiders, confirmed by qPCR analysis. We infer that dietary Cry1Ab expressing rice does not influence fecundity, nor expression levels of Vt-associated genes in P. pseudoannulata. Copyright © 2016. Published by Elsevier B.V.

Low-Light Dependence of the Magnetic Field Effect on Cryptochromes: Possible Relevance to Plant Ecology

PubMed Central

Vanderstraeten, Jacques; Gailly, Philippe; Malkemper, E. Pascal

2018-01-01

Various responses to static magnetic fields (MF) have been reported in plants, and it has been suggested that the geomagnetic field influences plant physiology. Accordingly, diverse mechanisms have been proposed to mediate MF effects in plants. The currently most probable sensor candidates are cryptochromes (Cry) which are sensitive to submillitesla MF. Here, we propose a quantitative approach of the MF effect on Cry depending on light intensity, and try to link it to a possible functional role for magnetic sensitivity in plants. Based on a theoretical evaluation and on a review of relevant data on Arabidopsis thaliana Cry 1, we point out that the MF effect on the signaling state of Cry, as well as the possible consequences of that effect on certain phenotypes (growth in particular) show parallel dependences on light intensity, being most prominent at low light levels. Based on these findings, we propose that Cry magnetosensitivity in plants could represent an ecological adaptation which regulates the amount of Cry signaling state under low light conditions. That hypothesis would preferentially be tested by studying sensitive and specific endpoints, such as the expression of clock proteins that are downregulated by Cry, but under light intensities lower than those used so far. Finally, we highlight that the low-light dependence of the MF effect described here could also apply to light-dependent functions of animal Cry, in particular magnetoreception which, from the present evaluation, would be based on the magnetic sensitivity of the photoreduction reaction, like in plants. PMID:29491873

Transcriptional analysis of Penaeus stylirostris densovirus genes

USDA-ARS?s Scientific Manuscript database

Penaeus stylirostris densovirus (PstDNV) genome contains three open reading frames (ORFs), left, middle, and right, which encode a non-structural (NS) protein, an unknown protein, and a capsid protein (CP), respectively. Transcription mapping revealed that P2, P11 and P61 promoters transcribe the le...

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

Treesearch

Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

2001-01-01

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

Effects of lymphatic drainage and cryotherapy on indirect markers of muscle damage.

PubMed

Behringer, Michael; Jedlicka, Diana; Mester, Joachim

2018-06-01

Muscle enzymes are cleared from the extracellular space by the lymphatic system, while smaller proteins enter the bloodstream directly. We investigated if manual lymphatic drainage (MLD), local cryotherapy (CRY), and rest (RST) differently affect the time course of creatine kinase (CK, 84 kDa) and heart-type fatty acid binding protein (h-FABP, 15 kDa) in the blood. Randomized controlled trial. After 4x20 unilateral, eccentric accentuated knee extensions (with one-third of the maximal isometric force) 30 sports students randomly received either a 30 min MLD, CRY or they rested (RST) for the same amount of time. CK, h-FABP, neutrophil granulocytes, and the perceived muscle soreness were assessed before, immediately after, and 1 hour, 4 hours, and 24 hours after the exercise. All measures increased significantly (P<0.001) after the protocol indicating that muscle damage was induced. However, the responses did not differ between the treatments. Large and small damage markers were not affected differently by MLD, CRY, or RST, when applied for 30 min and no beneficial effects on inflammation or muscle soreness could be found for MLD and CRY when compared to RST. This information is particularly important for those sports physicians and conditioning specialists who use biochemical muscle damage markers to adjust the training load and volume of athletes.

Molecular basis for blue light-dependent phosphorylation of Arabidopsis cryptochrome 2

PubMed Central

Liu, Qing; Wang, Qin; Deng, Weixian; Wang, Xu; Piao, Mingxin; Cai, Dawei; Li, Yaxing; Barshop, William D.; Yu, Xiaolan; Zhou, Tingting; Liu, Bin; Oka, Yoshito; Wohlschlegel, James; Zuo, Zecheng; Lin, Chentao

2017-01-01

Plant cryptochromes undergo blue light-dependent phosphorylation to regulate their activity and abundance, but the protein kinases that phosphorylate plant cryptochromes have remained unclear. Here we show that photoexcited Arabidopsis cryptochrome 2 (CRY2) is phosphorylated in vivo on as many as 24 different residues, including 7 major phosphoserines. We demonstrate that four closely related Photoregulatory Protein Kinases (previously referred to as MUT9-like kinases) interact with and phosphorylate photoexcited CRY2. Analyses of the ppk123 and ppk124 triple mutants and amiR4k artificial microRNA-expressing lines demonstrate that PPKs catalyse blue light-dependent CRY2 phosphorylation to both activate and destabilize the photoreceptor. Phenotypic analyses of these mutant lines indicate that PPKs may have additional substrates, including those involved in the phytochrome signal transduction pathway. These results reveal a mechanism underlying the co-action of cryptochromes and phytochromes to coordinate plant growth and development in response to different wavelengths of solar radiation in nature. PMID:28492234

Composition of forage and grain from second-generation insect-protected corn MON 89034 is equivalent to that of conventional corn (Zea mays L.).

PubMed

Drury, Suzanne M; Reynolds, Tracey L; Ridley, William P; Bogdanova, Natalia; Riordan, Susan; Nemeth, Margaret A; Sorbet, Roy; Trujillo, William A; Breeze, Matthew L

2008-06-25

Insect-protected corn hybrids containing Cry insecticidal proteins derived from Bacillus thuringiensis have protection from target pests and provide effective management of insect resistance. MON 89034 hybrids have been developed that produce both the Cry1A.105 and Cry2Ab2 proteins, which provide two independent modes of insecticidal action against the European corn borer ( Ostrinia nubilalis ) and other lepidopteran insect pests of corn. The composition of MON 89034 corn was compared to conventional corn by measuring proximates, fiber, and minerals in forage and by measuring proximates, fiber, amino acids, fatty acids, vitamins, minerals, antinutrients, and secondary metabolites in grain collected from 10 replicated field sites across the United States and Argentina during the 2004-2005 growing seasons. Analyses established that the forage and grain from MON 89034 are compositionally comparable to the control corn hybrid and conventional corn reference hybrids. These findings support the conclusion that MON 89034 is compositionally equivalent to conventional corn hybrids.

A Study of Gibberellin Homeostasis and Cryptochrome-Mediated Blue Light Inhibition of Hypocotyl Elongation1[W][OA

PubMed Central

Zhao, Xiaoying; Yu, Xuhong; Foo, Eloise; Symons, Gregory M.; Lopez, Javier; Bendehakkalu, Krishnaprasad T.; Xiang, Jing; Weller, James L.; Liu, Xuanming; Reid, James B.; Lin, Chentao

2007-01-01

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA4 in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA4 content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs. PMID:17644628

Rapid simulated gastric fluid digestion of in-seed/grain proteins expressed in genetically engineered crops.

PubMed

Schafer, Barry W; Embrey, Shawna K; Herman, Rod A

2016-11-01

The speed of simulated gastric digestion of proteins expressed in genetically engineered (GE) crops is commonly used to inform the allergenicity risk assessment. However, persistence of purified proteins in simulated gastric fluid (SGF) is poorly correlated with the allergenic status of proteins. It has been proposed that the plant or food matrix may affect the digestion of proteins and should be considered in interpreting digestion results. Here the SGF digestion of several GE proteins both as purified preparations and in soybean, corn, and cotton seed/grain extracts (in-matrix) are compared. Cry1F, Cry1Ac, phosphinothricin acetyltransferase (PAT), aryloxyalkanoate dioxygenase-1 (AAD-1), aryloxyalkanoate dioxygenase-12 (AAD-12), and double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) were all found to rapidly digest both as purified protein preparations and in seed/grain extracts from GE crops expressing these proteins. Based on these results, purified protein from microbial sources is a suitable surrogate for proteins in-matrix when conducting SGF digestion studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Resistance to Bacillus thuringiensis Mediated by an ABC Transporter Mutation Increases Susceptibility to Toxins from Other Bacteria in an Invasive Insect

PubMed Central

Zhang, Dandan; Gong, Lingling; He, Fei; Soberón, Mario; Bravo, Alejandra; Tabashnik, Bruce E.; Wu, Kongming

2016-01-01

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control. PMID:26872031

Comparative analysis of genomics and proteomics in Bacillus thuringiensis 4.0718.

PubMed

Rang, Jie; He, Hao; Wang, Ting; Ding, Xuezhi; Zuo, Mingxing; Quan, Meifang; Sun, Yunjun; Yu, Ziquan; Hu, Shengbiao; Xia, Liqiu

2015-01-01

Bacillus thuringiensis is a widely used biopesticide that produced various insecticidal active substances during its life cycle. Separation and purification of numerous insecticide active substances have been difficult because of the relatively short half-life of such substances. On the other hand, substances can be synthetized at different times during development, so samples at different stages have to be studied, further complicating the analysis. A dual genomic and proteomic approach would enhance our ability to identify such substances, and particularily using mass spectrometry-based proteomic methods. The comparative analysis for genomic and proteomic data have showed that not all of the products deduced from the annotated genome could be identified among the proteomic data. For instance, genome annotation results showed that 39 coding sequences in the whole genome were related to insect pathogenicity, including five cry genes. However, Cry2Ab, Cry1Ia, Cytotoxin K, Bacteriocin, Exoenzyme C3 and Alveolysin could not be detected in the proteomic data obtained. The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry. This analysis revealed Spo0A~P, SigF, SigE(+), SigK(+) and SigG(+), all known to play an important role in the process of spore formation regulatory network, also were displayed in the proteomic data. Through the comparison of the two data sets, it was possible to infer that some genes were silenced or were expressed at very low levels. For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons. With this comparative study a relatively complete database can be constructed and used to transform hereditary material, thereby prompting the high expression of toxic proteins. A theoretical basis is provided for constructing highly virulent engineered bacteria and for promoting the application of proteogenomics in the life sciences.

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton.

PubMed

Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

2017-01-01

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels.

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton

PubMed Central

Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

2017-01-01

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels. PMID:28099508

Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions.

PubMed

Breitler, Jean Christophe; Vassal, Jean Michel; del Mar Catala, Maria; Meynard, Donaldo; Marfà, Victoria; Melé, Enric; Royer, Monique; Murillo, Isabel; San Segundo, Blanca; Guiderdoni, Emmanuel; Messeguer, Joaquima

2004-09-01

Seven homozygous transgenic lines of two European commercial cultivars of rice (Ariete (A) and Senia (S)), harbouring the cry1B or cry1Aa Bacillus thuringiensis (Bt) delta-endotoxin genes, were field evaluated for protection from striped stem borer (SSB) (Chilo suppressalis) damage during the 2001 and 2002 summer crop seasons in the Delta de l'Ebre region, Spain. The plant codon-optimized toxin gene was placed under the control of the promoter of either the constitutive ubi1 gene or the wound-inducible mpi gene from maize. Stable, high-level, insecticidal protein accumulation was observed throughout root, leaf and seed tissues of field-grown plants harbouring the cry1B (lines A64.1, A33.1, A3.4 and S98.9) or cry1Aa (lines S05.1 and A19.14) genes under the control of the ubi1 promoter. Conversely, no toxin was detected in unwounded vegetative tissues of the A9.1 line harbouring the cry1B gene controlled by the mpi promoter, indicating that natural environmental stresses did not trigger the activity of the wound-inducible promoter. However, the toxin accumulated at 0.2% total soluble proteins in A9.1 sheath tissue exhibiting brown lesions resulting from SSB damage. The agronomical traits and performance of the transgenic lines were generally comparable with parental controls, except in the two lines accumulating Cry1Aa, which exhibited a high frequency of plants non-true to type. Natural infestation was assisted with manual infestations of L2/L3 SSB larvae in border control plants surrounding the experimental plots, which served as a reservoir for the second-cycle SSB population. The observation of damage (brown lesions and dead hearts) during the crop season and dissection of plants at harvest stage revealed a range of protection amongst the transgenic lines, which was highly consistent with the level of toxin accumulation and with previous experience in greenhouse assays. Lines A3.4 and S05.1 were found to exhibit stable and full protection against SSB attacks, mediated by the accumulation of Cry1B and Cry1Aa toxin, respectively, which was comparable with that afforded by the spraying of chemical insecticides on control plants. The wound-induced A9.1 line exhibited a satisfactory level of protection, with a notably low level of penetration of SSB larvae in the stems, but higher external symptoms than constitutive lines, probably due to the time lag to benefit from the protective effect of Cry1B.

A mathematical model of exposure of non-target Lepidoptera to Bt-maize pollen expressing Cry1Ab within Europe.

PubMed

Perry, J N; Devos, Y; Arpaia, S; Bartsch, D; Gathmann, A; Hails, R S; Kiss, J; Lheureux, K; Manachini, B; Mestdagh, S; Neemann, G; Ortego, F; Schiemann, J; Sweet, J B

2010-05-07

Genetically modified (GM) maize MON810 expresses a Cry1Ab insecticidal protein, derived from Bacillus thuringiensis (Bt), toxic to lepidopteran target pests such as Ostrinia nubilalis. An environmental risk to non-target Lepidoptera from this GM crop is exposure to harmful amounts of Bt-containing pollen deposited on host plants in or near MON810 fields. An 11-parameter mathematical model analysed exposure of larvae of three non-target species: the butterflies Inachis io (L.), Vanessa atalanta (L.) and moth Plutella xylostella (L.), in 11 representative maize cultivation regions in four European countries. A mortality-dose relationship was integrated with a dose-distance relationship to estimate mortality both within the maize MON810 crop and within the field margin at varying distances from the crop edge. Mortality estimates were adjusted to allow for physical effects; the lack of temporal coincidence between the susceptible larval stage concerned and the period over which maize MON810 pollen is shed; and seven further parameters concerned with maize agronomy and host-plant ecology. Sublethal effects were estimated and allowance made for aggregated pollen deposition. Estimated environmental impact was low: in all regions, the calculated mortality rate for worst-case scenarios was less than one individual in every 1572 for the butterflies and one in 392 for the moth.

A mathematical model of exposure of non-target Lepidoptera to Bt-maize pollen expressing Cry1Ab within Europe

PubMed Central

Perry, J. N.; Devos, Y.; Arpaia, S.; Bartsch, D.; Gathmann, A.; Hails, R. S.; Kiss, J.; Lheureux, K.; Manachini, B.; Mestdagh, S.; Neemann, G.; Ortego, F.; Schiemann, J.; Sweet, J. B.

2010-01-01

Genetically modified (GM) maize MON810 expresses a Cry1Ab insecticidal protein, derived from Bacillus thuringiensis (Bt), toxic to lepidopteran target pests such as Ostrinia nubilalis. An environmental risk to non-target Lepidoptera from this GM crop is exposure to harmful amounts of Bt-containing pollen deposited on host plants in or near MON810 fields. An 11-parameter mathematical model analysed exposure of larvae of three non-target species: the butterflies Inachis io (L.), Vanessa atalanta (L.) and moth Plutella xylostella (L.), in 11 representative maize cultivation regions in four European countries. A mortality–dose relationship was integrated with a dose–distance relationship to estimate mortality both within the maize MON810 crop and within the field margin at varying distances from the crop edge. Mortality estimates were adjusted to allow for physical effects; the lack of temporal coincidence between the susceptible larval stage concerned and the period over which maize MON810 pollen is shed; and seven further parameters concerned with maize agronomy and host-plant ecology. Sublethal effects were estimated and allowance made for aggregated pollen deposition. Estimated environmental impact was low: in all regions, the calculated mortality rate for worst-case scenarios was less than one individual in every 1572 for the butterflies and one in 392 for the moth. PMID:20053648

Pollen contamination in seed mixture increases the dominance of resistance to Bt maize in Spodoptera frugiperda (Lepidoptera: Noctuidae).

PubMed

Yang, Fei; Kerns, David L; Brown, Sebe; Head, Graham P; Huang, Fangneng

2017-11-01

Seed mixture, also called 'RIB', has been used to provide refuge populations for delaying insect resistance. Pollen contamination in RIB could result in refuge kernels of non-Bt maize expressing variable Bt proteins. Data are lacking regarding the impact of pollen contamination on evolution of resistance for ear-feeding insects. Here, we used Spodoptera frugiperda and Cry1F-maize as a model to examine if pollen contamination in RIB increases the dominance of insect resistance. Pollen contamination caused >66% refuge kernels in 5:95% (non-Bt:Bt) and 20:80% RIBs to express Cry1F protein. Survival at adult stage on pure non-Bt ears was similar (54.4-63.3%) among Cry1F-susceptible (SS), Cry1F-resistant (RR) and Cry1F-heterozygous (RS) S. frugiperda. On Bt ears, survival was similar between SS and RS (0.0-1.7%), but it was significantly less than that of RR (59.2%). On the two RIB refuge ears, survival at adult stage for RS (42.3% in 5:95% RIB; 50.0% in 20:80% RIB) was significantly higher than for SS (8.7% in 5:95% RIB; 10.0% in 20:80% RIB). The results suggest that pollen contamination in RIB could increase the dominance of resistance for ear-feeding insects by significantly reducing susceptible refuge populations and supporting higher survival of heterozygotes relative to homozygous susceptible insects. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Ancestral and derived protein import pathways in the mitochondrion of Reclinomonas americana.

PubMed

Tong, Janette; Dolezal, Pavel; Selkrig, Joel; Crawford, Simon; Simpson, Alastair G B; Noinaj, Nicholas; Buchanan, Susan K; Gabriel, Kipros; Lithgow, Trevor

2011-05-01

The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or "translocase in the inner mitochondrial membrane complex." Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.

Effects of 90-day feeding of transgenic Bt rice TT51 on the reproductive system in male rats.

PubMed

Wang, Er Hui; Yu, Zhou; Hu, Jing; Xu, Hai Bin

2013-12-01

Rice is a staple food crop; however, the threat of pests leads to a serious decline in its output and quality. The CryAb/CryAc gene, encodes a synthetic fusion Bacillus thuringiensis (Bt) crystal protein, was introduced into rice MingHui63 to produce insect-resistant rice TT51. This study was undertaken to investigate potential unintended effects of TT51 on the reproductive system in male rats. Male rats were treated with diets containing 60% of either TT51 or MingHui63 by weight, nutritionally balanced to an AIN93G diet, for 90days. An additional negative control group of rats were fed with a rice-based AIN93G diet. Body weights, food intake, hematology, serum chemistry, serum hormone levels, sperm parameters and relative organ/body weights were measured, and gross as well as microscopic pathology were examined. No diet-related significant differences in the values of response variables were observed between rats that were fed with diet containing transgenic TT51, MingHui63 and the control in this 90-day feeding study. In addition, necropsy and histopathology examination indicated no treatment-related changes. The results from the present study indicated that TT51 does not appear to exert any effect on the reproductive system in male rats compared with MingHui63 or the control. Copyright © 2013 Elsevier Ltd. All rights reserved.

Deletion of a Single-Copy Trna Affects Microtubule Function in Saccharomyces Cerevisiae

PubMed Central

Reijo, R. A.; Cho, D. S.; Huffaker, T. C.

1993-01-01

rts1-1 was identified as an extragenic suppressor of tub2-104, a cold-sensitive allele of the sole gene encoding β-tubulin in the yeast, Saccharomyces cerevisiae. In addition, rts1-1 cells are heat sensitive and resistant to the microtubule-destabilizing drug, benomyl. The rts1-1 mutation is a deletion of approximately 5 kb of genomic DNA on chromosome X that includes one open reading frame and three tRNA genes. Dissection of this region shows that heat sensitivity is due to deletion of the open reading frame (HIT1). Suppression and benomyl resistance are caused by deletion of the gene encoding a tRNA(AGG)(Arg) (HSX1). Northern analysis of rts1-1 cells indicates that HSX1 is the only gene encoding this tRNA. Deletion of HSX1 does not suppress the tub2-104 mutation by misreading at the AGG codons in TUB2. It also does not suppress by interfering with the protein arginylation that targets certain proteins for degradation. These results leave open the prospect that this tRNA(AGG)(Arg) plays a novel role in the cell. PMID:8307335

Mapping and Sequencing of the Canine NRAMP1 Gene and Identification of Mutations in Leishmaniasis-Susceptible Dogs

PubMed Central

Altet, Laura; Francino, Olga; Solano-Gallego, Laia; Renier, Corinne; Sánchez, Armand

2002-01-01

The NRAMP1 gene (Slc11a1) encodes an ion transporter protein involved in the control of intraphagosomal replication of parasites and in macrophage activation. It has been described in mice as the determinant of natural resistance or susceptibility to infection with antigenically unrelated pathogens, including Leishmania. Our aims were to sequence and map the canine Slc11a1 gene and to identify mutations that may be associated with resistance or susceptibility to Leishmania infection. The canine Slc11a1 gene has been mapped to dog chromosome CFA37 and covers 9 kb, including a 700-bp promoter region, 15 exons, and a polymorphic microsatellite in intron 1. It encodes a 547-amino-acid protein that has over 87% identity with the Slc11a1 proteins of different mammalian species. A case-control study with 33 resistant and 84 susceptible dogs showed an association between allele 145 of the microsatellite and susceptible dogs. Sequence variant analysis was performed by direct sequencing of the cDNA and the promoter region of four unrelated beagles experimentally infected with Leishmania infantum to search for possible functional mutations. Two of the dogs were classified as susceptible and the other two were classified as resistant based on their immune responses. Two important mutations were found in susceptible dogs: a G-rich region in the promoter that was common to both animals and a complete deletion of exon 11, which encodes the consensus transport motif of the protein, in the unique susceptible dog that needed an additional and prolonged treatment to avoid continuous relapses. A study with a larger dog population would be required to prove the association of these sequence variants with disease susceptibility. PMID:12010961

Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter.

PubMed

Miyake, Tsuyoshi; Hiraishi, Hiroyuki; Sammoto, Hiroyuki; Ono, Bun-Ichiro

2003-10-10

The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004-1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

Meta-analyses of the sensitivity and specificity of ante-mortem and post-mortem diagnostic tests for bovine tuberculosis in the UK and Ireland.

PubMed

Nuñez-Garcia, Javier; Downs, Sara H; Parry, Jessica E; Abernethy, Darrell A; Broughan, Jennifer M; Cameron, Angus R; Cook, Alasdair J; de la Rua-Domenech, Ricardo; Goodchild, Anthony V; Gunn, Jane; More, Simon J; Rhodes, Shelley; Rolfe, Simon; Sharp, Michael; Upton, Paul A; Vordermeier, H Martin; Watson, Eamon; Welsh, Michael; Whelan, Adam O; Woolliams, John A; Clifton-Hadley, Richard S; Greiner, Matthias

2018-05-01

Bovine Tuberculosis (bTB) in cattle is a global health problem and eradication of the disease requires accurate estimates of diagnostic test performance to optimize their efficiency. The objective of this study was, through statistical meta-analyses, to obtain estimates of sensitivity (Se) and specificity (Sp), for 14 different ante-mortem and post-mortem diagnostic tests for bTB in cattle. Using data from a systematic review of the scientific literature (published 1934-2009) diagnostic Se and Sp were estimated using Bayesian logistic regression models adjusting for confounding factors. Random effect terms were used to account for unexplained heterogeneity. Parameters in the models were implemented using Markov Chain Monte Carlo (MCMC), and posterior distributions for the diagnostic parameters with adjustment for covariates (confounding factors) were obtained using the inverse logit function. Estimates for Se and/or Sp of the tuberculin skin tests and the IFN-γ blood test were compared with estimates published 2010-2015. Median Se for the single intradermal comparative cervical tuberculin skin (SICCT) test (standard interpretation) was 0.50 and Bayesian credible intervals (CrI) were wide (95% CrI 0.26, 0.78). Median Sp for the SICCT test was 1.00 (95% CrI 0.99, 1.00). Estimates for the IFN-γ blood test Bovine Purified Protein Derivative (PPD)-Avian PPD and Early Secreted Antigen target 6 and Culture Filtrate Protein 10 (ESAT-6/CFP10) ESAT6/CFP10 were 0.67 (95% CrI 0.49, 0.82) and 0.78 (95% CrI 0.60, 0.90) respectively for Se, and 0.98 (95% CrI 0.96, 0.99) and 0.99 (95% CrI 0.99, 1.00) for Sp. The study provides an overview of the accuracy of a range of contemporary diagnostic tests for bTB in cattle. Better understanding of diagnostic test performance is essential for the design of effective control strategies and their evaluation. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

The characterization of electroplex generated from the interface between 2-(4-trifluoromethyl-2-hydroxyphenyl)benzothiazole] zinc and N,N'-diphenyl-N,N'- bis(1-naphthyl)-(1,1'-biphenyl)-4,4'-diamine

NASA Astrophysics Data System (ADS)

Zhang, Ye; Hao, Yuying; Meng, Weixin; Xu, Huixia; Wang, Hua; Xu, Bingshe

2012-03-01

The electroplex between (2-(4-trifluoromethyl-2-hydroxyphenyl)benzothiazole) zinc [Zn(4-TfmBTZ)2] as an electron-acceptor and N,N'-diphenyl-N,N'-bis(1-naphthyl)-(1,1'-biphenyl)-4,4'-diamine (NPB) as an electron-donor was characterized by bilayer, blend, and multilayer quantum-well (MQW) device, respectively. The blend composition and quantum-well number are effective parameters for tuning electroluminescence color. White light with high color purity and color rendering index (CRI) was observed from these devices based on Zn(4-TfmBTZ)2/NPB. Moreover, the blend and MQW devices all exhibit high operation stability, hence excellent color stability. For the device with 5 mol% NPB in blend layer, its Commission International Del'Eclairage (CIE) coordinate region is x=0.28-0.31, y=0.33-0.35 and CRI is 83.3-91.2 at 5-9 V. For MQW structure device with NPB of 60 nm thickness, its CIE coordinate region is x=0.29-0.32, y=0.31-0.34 and CRI=87.9-92.5 at 10-15 V. Such high color stability and purity and CRI, being close to ideal white light, are of current important for white OLED.

Toxin stability improvement and toxicity increase against dipteran and lepidopteran larvae of Bacillus thuringiensis crystal protein Cry2Aa.

PubMed

Elleuch, Jihen; Jaoua, Samir; Ginibre, Carole; Chandre, Fabrice; Tounsi, Slim; Zghal, Raida Z

2016-12-01

Bacillus thuringiensis δ-endotoxins are the most widely used biopesticides for controlling economically important crop pests and disease vectors. Improving their efficacy is of great benefit. Here, an improvement in Cry2Aa δ-endotoxin toxicity was attempted via a cry gene over expression system using P20 from B. thuringiensis israelensis. The coexpression of Cry2Aa with P20 resulted in a seven fold increase in its production yield in B. thuringiensis. Generated crystals proved to be significantly more toxic (505.207 µg g -1 , 1.99 mg L -1 and 1.49 mg L -1 ) than the P20-lacking control (720.78 µg g -1 , 705.69 mg L -1 and 508.51 mg L -1 ) against Ephestia kuehniella, Aedes aegypti and Culex pipiens larvae respectively. In vitro, processing experiments revealed a P20-mediated protection of Cry2Aa against degradation under larval gut conditions. Thus, P20 could promote the maintenance of a tightly packaged conformation of Cry2Aa toxins in the larval midgut upon correct activation and binding to its membrane receptors. Based on their resistance against excessive proteolysis, Cry2Aa δ-endotoxins, produced in the presence of P20, could be considered as a successful control agent for E. kuehniella and an effective alternative for mosquito control, implying its possible exploitation in pest management programmes. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Molecular identity and gene expression of aldosterone synthase cytochrome P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okamoto, Mitsuhiro; Nonaka, Yasuki; Takemori, Hiroshi

11{beta}-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11{beta}-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolatedmore » from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.« less

Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru.

PubMed

Vasilakis, Nikos; Castro-Llanos, Fanny; Widen, Steven G; Aguilar, Patricia V; Guzman, Hilda; Guevara, Carolina; Fernandez, Roberto; Auguste, Albert J; Wood, Thomas G; Popov, Vsevolod; Mundal, Kirk; Ghedin, Elodie; Kochel, Tadeusz J; Holmes, Edward C; Walker, Peter J; Tesh, Robert B

2014-04-01

Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.

Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru

PubMed Central

Castro-Llanos, Fanny; Widen, Steven G.; Aguilar, Patricia V.; Guzman, Hilda; Guevara, Carolina; Fernandez, Roberto; Auguste, Albert J.; Wood, Thomas G.; Popov, Vsevolod; Mundal, Kirk; Ghedin, Elodie; Kochel, Tadeusz J.; Holmes, Edward C.; Walker, Peter J.; Tesh, Robert B.

2014-01-01

Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family. PMID:24421116

GmHs1-1, encoding a calcineurin-like protein, controls hard-seededness in soybean

USDA-ARS?s Scientific Manuscript database

Loss of seed-coat impermeability was an essential step towards domestication of many leguminous crops for production of their highly nutritious seeds. Here we show that seed-coat impermeability in wild soybean is controlled by a single gene, Hard seededness 1 (Hs1), which encodes a calcineurin-like ...

Impact of transgenic soybean expressing Cry1Ac and Cry1F proteins on the non-target arthropod community associated with soybean in Brazil

PubMed Central

2018-01-01

Field-scale studies that examine the potential for adverse effects of Bt crop technology on non-target arthropods may supplement data from laboratory studies to support an environmental risk assessment. A three year field study was conducted in Brazil to evaluate potential for adverse effects of cultivating soybean event DAS-81419-2 that produces the Cry1Ac and Cry1F proteins. To do so, we examined the diversity and abundance of non-target arthropods (NTAs) in Bt soybean in comparison with its non-Bt near isoline, with and without conventional insecticide applications, in three Brazilian soybean producing regions. Non-target arthropod abundance was surveyed using Moericke traps (yellow pan) and pitfall trapping. Total abundance (N), richness (S), Shannon-Wiener (H’), Simpson’s (D) and Pielou’s evenness (J) values for arthropod samples were calculated for each treatment and sampling period (soybean growth stages). A faunistic analysis was used to select the most representative NTAs which were used to describe the NTA community structure associated with soybean, and to test for effects due to the treatments effects via application of the Principal Response Curve (PRC) method. Across all years and sites, a total of 254,054 individuals from 190 taxa were collected by Moericke traps, while 29,813 individuals from 100 taxa were collected using pitfall traps. Across sites and sampling dates, the abundance and diversity measurements of representative NTAs were not significantly affected by Bt soybean as compared with non-sprayed non-Bt soybean. Similarly, community analyses and repeated measures ANOVA, when applicable, indicated that neither Bt soybean nor insecticide sprays altered the structure of the NTA communities under study. These results support the conclusion that transgenic soybean event DAS-81419-2 producing Cry1Ac and Cry1F toxins does not adversely affect the NTA community associated with soybean. PMID:29394266

Do educational materials change knowledge and behaviour about crying and shaken baby syndrome? A randomized controlled trial.

PubMed

Barr, Ronald G; Barr, Marilyn; Fujiwara, Takeo; Conway, Jocelyn; Catherine, Nicole; Brant, Rollin

2009-03-31

Shaken baby syndrome often occurs after shaking in response to crying bouts. We questioned whether the use of the educational materials from the Period of PURPLE Crying program would change maternal knowledge and behaviour related to shaking. We performed a randomized controlled trial in which 1279 mothers received materials from the Period of PURPLE Crying program or control materials during a home visit by a nurse by 2 weeks after the birth of their child. At 5 weeks, the mothers completed a diary to record their behaviour and their infants' behaviour. Two months after giving birth, the mothers completed a telephone survey to assess their knowledge and behaviour. The mean score (range 0-100 points) for knowledge about infant crying was greater among mothers who received the PURPLE materials (63.8 points) than among mothers who received the control materials (58.4 points) (difference 5.4 points, 95% confidence interval [CI] 4.1 to 6.5 points). The mean scores were similar for both groups for shaking knowledge and reported maternal responses to crying, inconsolable crying and self-talk responses. Compared with mothers who received control materials, mothers who received the PURPLE materials reported sharing information about walking away if frustrated more often (51.5% v. 38.5%, difference 13.0%, 95% CI 6.9% to 19.2%), the dangers of shaking (49.3% v. 36.4%, difference 12.9%, 95% CI 6.8% to 19.0%), and infant crying (67.6% v. 60.0%, difference 7.6%, 95% CI 1.7% to 13.5%). Walking away during inconsolable crying was significantly higher among mothers who received the PURPLE materials than among those who received control materials (0.067 v. 0.039 events per day, rate ratio 1.7, 95% CI 1.1 to 2.6). The receipt of the Period of PURPLE Crying materials led to higher maternal scores for knowledge about infant crying and for some behaviours considered to be important for the prevention of shaking.

Do educational materials change knowledge and behaviour about crying and shaken baby syndrome? A randomized controlled trial

PubMed Central

Barr, Ronald G.; Barr, Marilyn; Fujiwara, Takeo; Conway, Jocelyn; Catherine, Nicole; Brant, Rollin

2009-01-01

Background Shaken baby syndrome often occurs after shaking in response to crying bouts. We questioned whether the use of the educational materials from the Period of PURPLE Crying program would change maternal knowledge and behaviour related to shaking. Methods We performed a randomized controlled trial in which 1279 mothers received materials from the Period of PURPLE Crying program or control materials during a home visit by a nurse by 2 weeks after the birth of their child. At 5 weeks, the mothers completed a diary to record their behaviour and their infants' behaviour. Two months after giving birth, the mothers completed a telephone survey to assess their knowledge and behaviour. Results The mean score (range 0–100 points) for knowledge about infant crying was greater among mothers who received the PURPLE materials (63.8 points) than among mothers who received the control materials (58.4 points) (difference 5.4 points, 95% confidence interval [CI] 4.1 to 6.5 points). The mean scores were similar for both groups for shaking knowledge and reported maternal responses to crying, inconsolable crying and self-talk responses. Compared with mothers who received control materials, mothers who received the PURPLE materials reported sharing information about walking away if frustrated more often (51.5% v. 38.5%, difference 13.0%, 95% CI 6.9% to 19.2%), the dangers of shaking (49.3% v. 36.4%, difference 12.9%, 95% CI 6.8% to 19.0%), and infant crying (67.6% v. 60.0%, difference 7.6%, 95% CI 1.7% to 13.5%). Walking away during inconsolable crying was significantly higher among mothers who received the PURPLE materials than among those who received control materials (0.067 v. 0.039 events per day, rate ratio 1.7, 95% CI 1.1 to 2.6). Interpretation The receipt of the Period of PURPLE Crying materials led to higher maternal scores for knowledge about infant crying and for some behaviours considered to be important for the prevention of shaking. (ClinicalTrials.gov trial register no. NCT00175422.) PMID:19255065

Intermittent bolus injection versus continuous infusion of furosemide in normal adult greyhound dogs.

PubMed

Adin, Darcy B; Taylor, Aaron W; Hill, Richard C; Scott, Karen C; Martin, Frank G

2003-01-01

Several studies in human subjects have demonstrated greater diuresis with constant rate infusion (CRI) furosemide than intermittent bolus (IB) furosemide. This study was conducted to compare the diuretic efficacy of the same total dose of IB furosemide and CRI furosemide in 6 healthy, adult Greyhound dogs in a randomized crossover design with a 2-week washout period between treatments. For IB administration, dogs received 3 mg/kg at 0 and 4 hours. For CRI administration, dogs received a 0.66 mg/kg loading dose followed by 0.66 mg/kg/h over 8 hours. The same volume of fluid was administered for both methods. Urine output was quantified hourly. Urine electrolyte concentrations, urine specific gravity (USG), packed cell volume (PCV), total protein (TP), serum electrolyte concentrations, total carbon dioxide (TCO2), serum creatinine (sCr), and blood urea nitrogen (BUN) were determined every 2 hours. Urine production and water intake were greater (P < or = 0.05) for CRI than IB. Urine sodium and calcium losses were greater (P < 0.05) and urine potassium loss was less (P = 0.03) for CRI than IB, but there was no evidence of a difference between methods for urine magnesium and chloride losses. Serum chloride concentration was less (P < 0.001), sCr concentration greater (P = 0.04). TP greater (P = 0.01), and PCV greater (P = 0.003) for CRI than IB. No differences in USG, TCO2, BUN, or serum potassium, sodium, and magnesium concentrations were detected between methods. The same total dose of CRI furosemide resulted in more diuresis, natriuresis, and calciuresis and less kaliuresis than IB furosemide in these normal Greyhound dogs over 8 hours, suggesting that furosemide is a more effective diuretic when administered by CRI than by IB.

An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity.

PubMed

Pacheco, Sabino; Gómez, Isabel; Sánchez, Jorge; García-Gómez, Blanca-Ines; Soberón, Mario; Bravo, Alejandra

2017-10-15

Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity. Copyright © 2017 American Society for Microbiology.

Many Saccharomyces cerevisiae Cell Wall Protein Encoding Genes Are Coregulated by Mss11, but Cellular Adhesion Phenotypes Appear Only Flo Protein Dependent.

PubMed

Bester, Michael C; Jacobson, Dan; Bauer, Florian F

2012-01-01

The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell-cell and cell-surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11Δ genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

PubMed

Masson, L; Moar, W J; van Frankenhuyzen, K; Bossé, M; Brousseau, R

1992-02-01

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.

Deregulation of Rab and Rab Effector Genes in Bladder Cancer

PubMed Central

Ho, Joel R.; Chapeaublanc, Elodie; Kirkwood, Lisa; Nicolle, Remy; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Southgate, Jennifer; Radvanyi, François; Goud, Bruno

2012-01-01

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer. PMID:22724020

Conventional and high-dose daunorubicin and idarubicin in acute myeloid leukaemia remission induction treatment: a mixed treatment comparison meta-analysis of 7258 patients.

PubMed

Sekine, Leo; Morais, Vinícius Daudt; Lima, Karine Margarites; Onsten, Tor Gunnar Hugo; Ziegelmann, Patrícia Klarmann; Ribeiro, Rodrigo Antonini

2015-12-01

Previous meta-analyses suggested that acute myeloid leukaemia induction regimens containing idarubicin (IDA) or high-dose daunorubicin (HDD) induce higher rates of complete remission (CR) than conventional-dose daunorubicin (CDD), with a possible benefit in overall survival. However, robust comparisons between these regimens are still lacking. We conducted a mixed treatment comparison meta-analysis regarding these three regimens. Mixed treatment comparison is a statistical method of data summarization that aggregates data from both direct and indirect effect estimates. Literature search strategy included MEDLINE, EMBASE, Cochrane, Scielo and LILACS, from inception until August 2013 and resulted in the inclusion of 17 trials enrolling 7258 adult patients. HDD [relative risk (RR) 1.13; 95% credible interval (CrI) 1.02-1.26] and IDA (RR 1.13; 95% CrI 1.05-1.23) showed higher CR rates than CDD. IDA also led to lower long-term overall mortality rates when compared with CDD (RR 0.93, 95% CrI 0.86-0.99), whereas HDD and CDD were no different (RR 0.94, 95% CrI 0.85-1.02). HDD and IDA comparison did not reach statistically significant differences in CR (RR 1.00; 95% CrI 0.89-1.11) and in long-term mortality (RR 1.01, 95% CrI 0.91-1.11). IDA and HDD are consistently superior to CDD in inducing CR, and IDA was associated with lower long-term mortality. On the basis of these findings, we recommend incorporation of IDA and HDD instead of the traditional CDD as standard treatments for acute myeloid leukaemia induction. The lack of HDD benefit on mortality, when compared with CDD in this study, should be cautiously addressed, because it may have been susceptible to underestimation because of statistical power limitations. Copyright © 2014 John Wiley & Sons, Ltd.

Feedback repression is required for mammalian circadian clock function.

PubMed

Sato, Trey K; Yamada, Rikuhiro G; Ukai, Hideki; Baggs, Julie E; Miraglia, Loren J; Kobayashi, Tetsuya J; Welsh, David K; Kay, Steve A; Ueda, Hiroki R; Hogenesch, John B

2006-03-01

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.

Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests.

PubMed

Betz, F S; Hammond, B G; Fuchs, R L

2000-10-01

Plants modified to express insecticidal proteins from Bacillus thuringiensis (referred to as Bt-protected plants) provide a safe and highly effective method of insect control. Bt-protected corn, cotton, and potato were introduced into the United States in 1995/1996 and grown on a total of approximately 10 million acres in 1997, 20 million acres in 1998, and 29 million acres globally in 1999. The extremely rapid adoption of these Bt-protected crops demonstrates the outstanding grower satisfaction of the performance and value of these products. These crops provide highly effective control of major insect pests such as the European corn borer, southwestern corn borer, tobacco budworm, cotton bollworm, pink bollworm, and Colorado potato beetle and reduce reliance on conventional chemical pesticides. They have provided notably higher yields in cotton and corn. The estimated total net savings to the grower using Bt-protected cotton in the United States was approximately $92 million in 1998. Other benefits of these crops include reduced levels of the fungal toxin fumonisin in corn and the opportunity for supplemental pest control by beneficial insects due to the reduced use of broad-spectrum insecticides. Insect resistance management plans are being implemented to ensure the prolonged effectiveness of these products. Extensive testing of Bt-protected crops has been conducted which establishes the safety of these products to humans, animals, and the environment. Acute, subchronic, and chronic toxicology studies conducted over the past 40 years establish the safety of the microbial Bt products, including their expressed insecticidal (Cry) proteins, which are fully approved for marketing. Mammalian toxicology and digestive fate studies, which have been conducted with the proteins produced in the currently approved Bt-protected plant products, have confirmed that these Cry proteins are nontoxic to humans and pose no significant concern for allergenicity. Food and feed derived from Bt-protected crops which have been fully approved by regulatory agencies have been shown to be substantially equivalent to the food and feed derived from conventional crops. Nontarget organisms exposed to high levels of Cry protein are virtually unaffected, except for certain insects that are closely related to the target pests. Because the Cry protein is contained within the plant (in microgram quantities), the potential for exposure to farm workers and nontarget organisms is extremely low. The Cry proteins produced in Bt-protected crops have been shown to rapidly degrade when crop residue is incorporated into the soil. Thus the environmental impact of these crops is negligible. The human and environmental safety of Bt-protected crops is further supported by the long history of safe use for Bt microbial pesticides around the world. Copyright 2000 Academic Press.

A Single Point Mutation Resulting in Cadherin Mislocalization Underpins Resistance against Bacillus thuringiensis Toxin in Cotton Bollworm*

PubMed Central

Xiao, Yutao; Dai, Qing; Hu, Ruqin; Pacheco, Sabino; Yang, Yongbo; Liang, Gemei; Soberón, Mario

2017-01-01

Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Resistance to B. thuringiensis toxins threatens this technology, and although different resistance mechanisms have been identified, some have not been completely elucidated. To gain new insights into these mechanisms, we performed multiple back-crossing from a 3000-fold Cry1Ac-resistant BtR strain from cotton bollworm (Helicoverpa armigera), isolating a 516-fold Cry1Ac-resistant strain (96CAD). Cry1Ac resistance in 96CAD was tightly linked to a mutant cadherin allele (mHaCad) that contained 35 amino acid substitutions compared with HaCad from a susceptible strain (96S). We observed significantly reduced levels of the mHaCad protein on the surface of the midgut epithelium in 96CAD as compared with 96S. Expression of both cadherin alleles from 96CAD and 96S in insect cells and immunofluorescence localization in insect midgut tissue sections showed that the HaCAD protein from 96S localizes on the cell membrane, whereas the mutant 96CAD-mHaCad was retained in the endoplasmic reticulum (ER). Mapping of the mutations identified a D172G substitution mainly responsible for cadherin mislocalization. Our finding of a mutation affecting membrane receptor trafficking represents an unusual and previously unrecognized B. thuringiensis resistance mechanism. PMID:28082675

Heat shock factor 1 induces crystallin-αB to protect against cisplatin nephrotoxicity

PubMed Central

Lou, Qiang; Hu, Yanzhong; Ma, Yuanfang

2016-01-01

Cisplatin, a wildly used chemotherapy drug, induces nephrotoxicity that is characterized by renal tubular cell apoptosis. In response to toxicity, tubular cells can activate cytoprotective mechanisms, such as the heat shock response. However, the role and regulation of the heat shock response in cisplatin-induced nephrotoxicity remain largely unclear. In the present study, we demonstrated the induction of heat shock factor (Hsf)1 and the small heat shock protein crystallin-αB (CryAB) during cisplatin nephrotoxicity in mice. Consistently, cisplatin induced Hsf1 and CryAB in a cultured renal proximal tubular cells (RPTCs). RPTCs underwent apoptosis during cisplatin treatment, which was increased when Hsf1 was knocked down. Transfection or restoration of Hsf1 into Hsf1 knockdown cells suppressed cisplatin-induced apoptosis, further supporting a cytoprotective role of Hsf1 and its associated heat shock response. Moreover, Hsf1 knockdown increased Bax translocation to mitochondria and cytochrome c release into the cytosol. In RPTCs, Hsf1 knockdown led to a specific downregulation of CryAB. Transfection of CryAB into Hsf1 knockdown cells diminished their sensitivity to cisplatin-induced apoptosis, suggesting that CryAB may be a key mediator of the cytoprotective effect of Hsf1. Taken together, these results demonstrate a heat shock response in cisplatin nephrotoxicity that is mediated by Hsf1 and CryAB to protect tubular cells against apoptosis. PMID:27194715

Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein.

PubMed

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-06-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

Characterization of the Tupaia Rhabdovirus Genome Reveals a Long Open Reading Frame Overlapping with P and a Novel Gene Encoding a Small Hydrophobic Protein

PubMed Central

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-01-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses. PMID:15890917

Mucosal and systemic adjuvant effects of cholera toxin and Cry1Ac protoxin on the specific antibody response to HIV-1 C4/V3 peptides are different and depend on the antigen co-administered.

PubMed

Esquivel-Pérez, Rita; Moreno-Fierros, Leticia

2005-01-01

Evidence from several sources support the assertion that cholera toxin (CT) is a potent immunogen and mucosal adjuvant; however there are also reports showing its lack of adjuvanticity against some antigens. Cry1Ac protoxin also exerts adjuvant effects in the antibody responses to proteins and polysaccharides but its adjuvanticity with regard to peptide vaccines had not been tested. To probe whether the adjuvant effects of these proteins varied depending on the antigen co-administered, we evaluated antipeptide antibody responses in serum and mucosal samples (vaginal, intestinal, and pulmonary) of mice that were immunized by intranasal or intraperitoneal routes with one of two distinct hybrid C4/V3 HIV peptides, either alone or with CT or Cry1Ac. The tested HIV 1 peptides differed in two aminoacids, T1SP10MN(A) was modified at the SP10 region by the substitution of the isoleucines 12 and 14 for cysteines and was called modified (m)T1SP10MN(A). Our data indicate that the adjuvant effects of CT and Cry1Ac are different. In addition they vary depending on the antigen co-administered and the number of antigen doses, because after three doses moderate adjuvant effects of CT and Cry1Ac on anti-peptide serum and mucosal antibody responses were observed only against the mT1SP10MN(A). In contrast, to attain significant adjuvant effects against the T1SP10MN, four doses were required. Interestingly we found that modification of the HIV peptide increases its immunogenicity without altering the principal neutralizing determinant (SP10).

Blue Light–Dependent Interaction between Cryptochrome2 and CIB1 Regulates Transcription and Leaf Senescence in Soybean[W

PubMed Central

Meng, Yingying; Li, Hongyu; Wang, Qin; Liu, Bin; Lin, Chentao

2013-01-01

Cryptochromes are blue light receptors that regulate light responses in plants, including various crops. The molecular mechanism of plant cryptochromes has been extensively investigated in Arabidopsis thaliana, but it has not been reported in any crop species. Here, we report a study of the mechanism of soybean (Glycine max) cryptochrome2 (CRY2a). We found that CRY2a regulates leaf senescence, which is a life history trait regulated by light and photoperiods via previously unknown mechanisms. We show that CRY2a undergoes blue light–dependent interaction with the soybean basic helix-loop-helix transcription activator CIB1 (for cryptochrome-interacting bHLH1) that specifically interacts with the E-box (CANNTG) DNA sequences. Analyses of transgenic soybean plants expressing an elevated or reduced level of the CRY2a or CIB1 demonstrate that CIB1 promotes leaf senescence, whereas CRY2a suppresses leaf senescence. Results of the gene expression and molecular interaction analyses support the hypothesis that CIB1 activates transcription of senescence-associated genes, such as WRKY DNA BINDING PROTEIN53b (WRKY53b), and leaf senescence. CIB1 interacts with the E-box–containing promoter sequences of the WRKY53b chromatin, whereas photoexcited CRY2a interacts with CIB1 to inhibit its DNA binding activity. These findings argue that CIB-dependent transcriptional regulation is an evolutionarily conserved CRY-signaling mechanism in plants, and this mechanism is opted in evolution to mediate light regulation of different aspects of plant development in different plant species. PMID:24272488

Cloning and characterization of a novel zinc finger gene in Xp11.2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Derry, J.M.J.; Jess, U.; Francke, U.

1995-11-20

During a systematic search for open reading frames in chromosome band Xp11.2, a novel gene (ZNF157) that encodes a putative 506-amino-acid protein with the sequence characteristics of a zinc-finger-containing transcription factor was isolated. ZNF157 is encoded by four exons distributed over >20 kb of genomic DNA. The second and third exons contain sequences similar to those of the previously described KRAB-A and KRAB-B domains, motifs that have been shown to mediate transcriptional repression in other members of the protein family. A fourth exon contains 12 zinc finger DNA binding motifs and finger linking regions characteristic of ZNF proteins of themore » Krueppel family. ZNF157 maps to the telomeric end of a cluster of ZNF genes that includes ZNF21, ZNF41, and ZNF81. 19 refs., 2 figs.« less

Genetic and biochemical characterization of field-evolved resistance to Bacillus thuringiensis toxin Cry1Ac in the diamondback moth, Plutella xylostella.

PubMed

Sayyed, Ali H; Raymond, Ben; Ibiza-Palacios, M Sales; Escriche, Baltasar; Wright, Denis J

2004-12-01

The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F1 progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.

INSECTICIDAL TOXIN FROM BACILLUS THURINGIENSIS IS RELEASED FROM ROOTS OF TRANSGENIC BT CORN IN VITRO AND IN SITU. (R826107)

EPA Science Inventory

Abstract

The insecticidal toxin encoded by the cry1Ab gene from Bacillus thuringiensis was released in root exudates from transgenic Bt corn during 40 days of growth in soil amended to 0, 3, 6, 9, or 12% (v/v) with montmorillonite or kaolinite in a...

Clock genes and salt-sensitive hypertension: a new type of aldosterone-synthesizing enzyme controlled by the circadian clock and angiotensin II.

PubMed

Okamura, Hitoshi; Doi, Masao; Goto, Kaoru; Kojima, Rika

2016-10-01

With the current societal norm of shiftwork and long working hours, maintaining a stable daily life is becoming very difficult. An irregular lifestyle disrupts circadian rhythms, resulting in the malfunction of body physiology and ultimately leading to lifestyle-related diseases, including hypertension. By analyzing completely arrhythmic Cry1/Cry2 double-knockout (Cry-null) mice, we found salt-sensitive hypertension accompanied by hyperaldosteronism. On the basis of a DNA microarray analysis of the adrenal gland and subsequent biochemical analyses, we discovered that Hsd3b6/HSD3B1, a subtype of 3β-HSD, is markedly overexpressed in aldosterone-producing cells in the Cry-null adrenal cortex. In addition, we found that Hsd3b6/HSD3B1, which converts pregnenolone to progesterone, is a clock-controlled gene and might also be a key enzyme for the regulation of aldosterone biosynthesis, in addition to the previously established CYP11B2, which synthesizes aldosterone from deoxycorticosterone. Importantly, angiotensin II induces HSD3B1 via the transcription factor NGFIB in human adrenocortical H295R cells, similarly to CYP11B2. As HSD3B1 levels are abnormally high in the adrenal aldosterone-producing cells of idiopathic hyperaldosteronism (IHA), the temporal component of this system in the pathophysiology of IHA is a promising area for future research.

Housing conditions associated with recurrent gastrointestinal infection in urban Aboriginal children in NSW, Australia: findings from SEARCH.

PubMed

Andersen, Melanie J; Skinner, Adam; Williamson, Anna B; Fernando, Peter; Wright, Darryl

2018-06-01

To examine the associations between housing and gastrointestinal infection in Aboriginal children in urban New South Wales. A total of 1,398 Aboriginal children were recruited through four Aboriginal Community Controlled Health Services. Multilevel regression modelling of survey data estimated associations between housing conditions and recurrent gastrointestinal infection, adjusting for sociodemographic and health factors. Of the sample, 157 children (11%) had recurrent gastrointestinal infection ever and 37 (2.7%) required treatment for recurrent gastrointestinal infection in the past month. Children in homes with 3+ housing problems were 2.51 (95% CrI 1.10, 2.49) times as likely to have recurrent gastrointestinal infection ever and 6.79 (95% CrI 2.11, 30.17) times as likely to have received recent treatment for it (versus 0-2 problems). For every additional housing problem, the prevalence of recurrent gastrointestinal infection ever increased by a factor of 1.28 (95% CrI 1.14, 1.47) and the prevalence of receiving treatment for gastrointestinal infection in the past month increased by a factor of 1.64 (95% CrI 1.20, 2.48). Housing problems were independently associated with recurrent gastrointestinal infection in a dose-dependent manner. Implications for public health: The role of housing as a potential determinant of health in urban Aboriginal children merits further attention in research and policy settings. © 2018 The Authors.

Plasma and serum concentrations of cytarabine administered via continuous intravenous infusion to dogs with meningoencephalomyelitis of unknown etiology.

PubMed

Early, P J; Crook, K I; Williams, L M; Davis, E G; Muñana, K R; Papich, M G; Messenger, K M

2017-08-01

The objective of this study was to evaluate the plasma and serum concentrations of cytarabine (CA) administered via constant rate infusion (CRI) in dogs with meningoencephalomyelitis of unknown etiology (MUE). Nineteen client-owned dogs received a CRI of CA at a dose of 25 mg/m 2 /h for 8 h as treatment for MUE. Dogs were divided into four groups, those receiving CA alone and those receiving CA in conjunction with other drugs. Blood samples were collected at 0, 1, 8, and 12 h after initiating the CRI. Plasma (n = 13) and serum (n = 11) cytarabine concentrations were measured by high-pressure liquid chromatography. The mean peak concentration (C MAX ) and area under the curve (AUC) after CRI administration were 1.70 ± 0.66 μg/mL and 11.39 ± 3.37 h·μg/mL, respectively, for dogs receiving cytarabine alone, 2.36 ± 0.35 μg/mL and 16.91 + 3.60 h·μg/mL for dogs administered cytarabine and concurrently on other drugs. Mean concentrations for all dogs were above 1.0 μg/mL at both the 1- and 8-h time points. The steady-state achieved with cytarabine CRI produces a consistent and prolonged exposure in plasma and serum, which is likely to produce equilibrium between blood and the central nervous system in dogs with a clinical diagnosis of MUE. Other medications commonly used to treat MUE do not appear to alter CA concentrations in serum and plasma. © 2016 John Wiley & Sons Ltd.

Accuracy of clinical diagnosis of Parkinson disease: A systematic review and meta-analysis.

PubMed

Rizzo, Giovanni; Copetti, Massimiliano; Arcuti, Simona; Martino, Davide; Fontana, Andrea; Logroscino, Giancarlo

2016-02-09

To evaluate the diagnostic accuracy of clinical diagnosis of Parkinson disease (PD) reported in the last 25 years by a systematic review and meta-analysis. We searched for articles published between 1988 and August 2014. Studies were included if reporting diagnostic parameters regarding clinical diagnosis of PD or crude data. The selected studies were subclassified based on different study setting, type of test diagnosis, and gold standard. Bayesian meta-analyses of available data were performed. We selected 20 studies, including 11 using pathologic examination as gold standard. Considering only these 11 studies, the pooled diagnostic accuracy was 80.6% (95% credible interval [CrI] 75.2%-85.3%). Accuracy was 73.8% (95% CrI 67.8%-79.6%) for clinical diagnosis performed mainly by nonexperts. Accuracy of clinical diagnosis performed by movement disorders experts rose from 79.6% (95% CrI 46%-95.1%) of initial assessment to 83.9% (95% CrI 69.7%-92.6%) of refined diagnosis after follow-up. Using UK Parkinson's Disease Society Brain Bank Research Center criteria, the pooled diagnostic accuracy was 82.7% (95% CrI 62.6%-93%). The overall validity of clinical diagnosis of PD is not satisfying. The accuracy did not significantly improve in the last 25 years, particularly in the early stages of disease, where response to dopaminergic treatment is less defined and hallmarks of alternative diagnoses such as atypical parkinsonism may not have emerged. Misclassification rate should be considered to calculate the sample size both in observational studies and randomized controlled trials. Imaging and biomarkers are urgently needed to improve the accuracy of clinical diagnosis in vivo. © 2016 American Academy of Neurology.

Association of Pharmacological Treatments for Obesity With Weight Loss and Adverse Events

PubMed Central

Khera, Rohan; Murad, Mohammad Hassan; Chandar, Apoorva K.; Dulai, Parambir S.; Wang, Zhen; Prokop, Larry J.; Loomba, Rohit; Camilleri, Michael; Singh, Siddharth

2017-01-01

IMPORTANCE Five medications have been approved for the management of obesity, but data on comparative effectiveness are limited. OBJECTIVE To compare weight loss and adverse events among drug treatments for obesity using a systematic review and network meta-analysis. DATA SOURCES MEDLINE, EMBASE, Web of Science, Scopus, and Cochrane Central from inception to March 23, 2016; clinical trial registries. STUDY SELECTION Randomized clinical trials conducted among overweight and obese adults treated with US Food and Drug Administration–approved long-term weight loss agents (orlistat, lorcaserin, naltrexone-bupropion, phentermine-topiramate, or liraglutide) for at least 1 year compared with another active agent or placebo. DATA EXTRACTION AND SYNTHESIS Two investigators identified studies and independently abstracted data using a predefined protocol. A Bayesian network meta-analysis was performed and relative ranking of agents was assessed using surface under the cumulative ranking (SUCRA) probabilities. Quality of evidence was assessed using GRADE criteria. MAIN OUTCOMES AND MEASURES Proportions of patients with at least 5%weight loss and at least 10% weight loss, magnitude of decrease in weight, and discontinuation of therapy because of adverse events at 1 year. RESULTS Twenty-eight randomized clinical trials with 29018 patients (median age, 46 years; 74%women; median baseline body weight, 100.5 kg; median baseline body mass index, 36.1) were included. A median 23%of placebo participants had at least 5%weight loss vs 75%of participants taking phentermine-topiramate (odds ratio [OR], 9.22; 95%credible interval [CrI], 6.63–12.85; SUCRA, 0.95), 63%of participants taking liraglutide (OR, 5.54; 95%CrI, 4.16–7.78; SUCRA, 0.83), 55%taking naltrexone-bupropion (OR, 3.96; 95%CrI, 3.03–5.11; SUCRA, 0.60), 49%taking lorcaserin (OR, 3.10; 95%CrI, 2.38–4.05; SUCRA, 0.39), and 44%taking orlistat (OR, 2.70; 95%CrI, 2.34–3.09; SUCRA, 0.22). All active agents were associated with significant excess weight loss compared with placebo at 1 year—phentermine-topiramate, 8.8 kg (95%CrI, −10.20 to −7.42 kg); liraglutide, 5.3 kg (95%CrI, −6.06 to −4.52 kg); naltrexone-bupropion, 5.0 kg (95%CrI, −5.94 to −3.96 kg); lorcaserin, 3.2 kg (95%CrI, −3.97 to −2.46 kg); and orlistat, 2.6 kg (95%CrI, −3.04 to −2.16 kg). Compared with placebo, liraglutide (OR, 2.95; 95%CrI, 2.11–4.23) and naltrexone-bupropion (OR, 2.64; 95%CrI, 2.10–3.35) were associated with the highest odds of adverse event–related treatment discontinuation. High attrition rates (30%–45%in all trials) were associated with lower confidence in estimates. CONCLUSIONS AND RELEVANCE Among overweight or obese adults, orlistat, lorcaserin, naltrexone-bupropion, phentermine-topiramate, and liraglutide, compared with placebo, were each associated with achieving at least 5%weight loss at 52 weeks. Phentermine-topiramate and liraglutide were associated with the highest odds of achieving at least 5%weight loss. PMID:27299618

Protein aggregation studied by forward light scattering and light transmission analysis

NASA Astrophysics Data System (ADS)

Penzkofer, A.; Shirdel, J.; Zirak, P.; Breitkreuz, H.; Wolf, E.

2007-12-01

The aggregation of the circadian blue-light photo-receptor cryptochrome from Drosophila melanogaster (dCry) is studied by transmission and forward light scattering measurement in the protein transparent wavelength region. The light scattering in forward direction is caused by Rayleigh scattering which is proportional to the degree of aggregation. The light transmission through the samples in the transparent region is reduced by Mie light scattering in all directions. It depends on the degree of aggregation and the monomer volume fill factor of the aggregates (less total scattering with decreasing monomer volume fill factor of protein globule) allowing a distinction between tightly packed protein aggregation (monomer volume fill factor 1) and loosely packed protein aggregation (monomer volume fill factor less than 1). An increase in aggregation with temperature, concentration, and blue-light exposure is observed. At a temperature of 4 °C and a protein concentration of less than 0.135 mM no dCry aggregation was observed, while at 24 °C and 0.327 mM gelation occurred (loosely packed aggregates occupying the whole solution volume).

Circadian pacemaking in cells and circuits of the suprachiasmatic nucleus.

PubMed

Hastings, M H; Brancaccio, M; Maywood, E S

2014-01-01

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker of the brain. It co-ordinates the daily rhythms of sleep and wakefulness, as well as physiology and behaviour, that set the tempo to our lives. Disturbance of this daily pattern, most acutely with jet-lag but more insidiously with rotational shift-work, can have severely deleterious effects for mental function and long-term health. The present review considers recent developments in our understanding of the properties of the SCN that make it a robust circadian time-keeper. It first focuses on the intracellular transcriptional/ translational feedback loops (TTFL) that constitute the cellular clockwork of the SCN neurone. Daily timing by these loops pivots around the negative regulation of the Period (Per) and Cryptochrome (Cry) genes by their protein products. The period of the circadian cycle is set by the relative stability of Per and Cry proteins, and this can be controlled by both genetic and pharmacological interventions. It then considers the function of these feedback loops in the context of cytosolic signalling by cAMP and intracellular calcium ([Ca(2+) ]i ), which are both outputs from, and inputs to, the TTFL, as well as the critical role of vasoactive intestinal peptide (VIP) signalling in synchronising cellular clocks across the SCN. Synchronisation by VIP in the SCN is paracrine, operating over an unconventionally long time frame (i.e. 24 h) and wide spatial domain, mediated via the cytosolic pathways upstream of the TTFL. Finally, we show how intersectional pharmacogenetics can be used to control G-protein-coupled signalling in individual SCN neurones, and how manipulation of Gq/[Ca(2+) ]i -signalling in VIP neurones can re-programme the circuit-level encoding of circadian time. Circadian pacemaking in the SCN therefore provides an unrivalled context in which to understand how a complex, adaptive behaviour can be organised by the dynamic activity of a relatively few gene products, operating in a clearly defined neuronal circuit, with both cell-autonomous and emergent, circuit-level properties. © 2014 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of The British Society for Neuroendocrinology.

Resistance Risk Assessment of Spodoptera frugiperda (Lepidoptera: Noctuidae) and Diatraea saccharalis (Lepidoptera: Crambidae) to Vip3Aa20 Insecticidal Protein Expressed in Corn.

PubMed

Bernardi, Oderlei; Bernardi, Daniel; Amado, Douglas; Sousa, Renan S; Fatoretto, Julio; Medeiros, Fernanda C L; Conville, Jared; Burd, Tony; Omoto, Celso

2015-12-01

Transgenic Agrisure Viptera 3 corn that expresses Cry1Ab, Vip3Aa20, and EPSPS proteins and Agrisure Viptera expressing Vip3Aa20 are used for control of Spodoptera frugiperda (J.E. Smith) and Diatraea saccharalis (F.) in Brazil. To support a resistance management program, resistance risk assessment studies were conducted to characterize the dose expression of Vip3Aa20 protein and level of control against these species. The Vip3Aa20 expression in Agrisure Viptera 3 and Agrisure Viptera decreased from V6 to V10 stage of growth. However, Vip3Aa20 expression in Agrisure Viptera 3 at V6 and V10 stages was 13- and 16-fold greater than Cry1Ab, respectively. The Vip3Aa20 expression in lyophilized tissue of Agrisure Viptera 3 and Agrisure Viptera diluted 25-fold in an artificial diet caused complete larval mortality of S. frugiperda and D. saccharalis. In contrast, lyophilized tissue of Bt11 at the same dilution does not provide complete mortality of these species. Agrisure Viptera 3 and Agrisure Viptera also caused a high level of mortality against S. frugiperda and D. saccharalis. Moreover, 100% mortality was observed for S. frugiperda larvae (neonates through fifth-instar larvae) when fed in corn with the Vip trait technology. Viptera corn achieves a high level of control against S. frugiperda and D. saccharalis providing a high dose, which is an important determination to support the refuge strategy for an effective resistance management program. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Bacterial bioluminescence regulates expression of a host cryptochrome gene in the squid-Vibrio symbiosis.

PubMed

Heath-Heckman, Elizabeth A C; Peyer, Suzanne M; Whistler, Cheryl A; Apicella, Michael A; Goldman, William E; McFall-Ngai, Margaret J

2013-04-02

The symbiosis between the squid Euprymna scolopes and its luminous symbiont, Vibrio fischeri, is characterized by daily transcriptional rhythms in both partners and daily fluctuations in symbiont luminescence. In this study, we sought to determine whether symbionts affect host transcriptional rhythms. We identified two transcripts in host tissues (E. scolopes cry1 [escry1] and escry2) that encode cryptochromes, proteins that influence circadian rhythms in other systems. Both genes cycled daily in the head of the squid, with a pattern similar to that of other animals, in which expression of certain cry genes is entrained by environmental light. In contrast, escry1 expression cycled in the symbiont-colonized light organ with 8-fold upregulation coincident with the rhythms of bacterial luminescence, which are offset from the day/night light regime. Colonization of the juvenile light organ by symbionts was required for induction of escry1 cycling. Further, analysis with a mutant strain defective in light production showed that symbiont luminescence is essential for cycling of escry1; this defect could be complemented by presentation of exogenous blue light. However, blue-light exposure alone did not induce cycling in nonsymbiotic animals, but addition of molecules of the symbiont cell envelope to light-exposed animals did recover significant cycling activity, showing that light acts in synergy with other symbiont features to induce cycling. While symbiont luminescence may be a character specific to rhythms of the squid-vibrio association, resident microbial partners could similarly influence well-documented daily rhythms in other systems, such as the mammalian gut.

Bacterial Bioluminescence Regulates Expression of a Host Cryptochrome Gene in the Squid-Vibrio Symbiosis

PubMed Central

Heath-Heckman, Elizabeth A. C.; Peyer, Suzanne M.; Whistler, Cheryl A.; Apicella, Michael A.; Goldman, William E.; McFall-Ngai, Margaret J.

2013-01-01

ABSTRACT The symbiosis between the squid Euprymna scolopes and its luminous symbiont, Vibrio fischeri, is characterized by daily transcriptional rhythms in both partners and daily fluctuations in symbiont luminescence. In this study, we sought to determine whether symbionts affect host transcriptional rhythms. We identified two transcripts in host tissues (E. scolopes cry1 [escry1] and escry2) that encode cryptochromes, proteins that influence circadian rhythms in other systems. Both genes cycled daily in the head of the squid, with a pattern similar to that of other animals, in which expression of certain cry genes is entrained by environmental light. In contrast, escry1 expression cycled in the symbiont-colonized light organ with 8-fold upregulation coincident with the rhythms of bacterial luminescence, which are offset from the day/night light regime. Colonization of the juvenile light organ by symbionts was required for induction of escry1 cycling. Further, analysis with a mutant strain defective in light production showed that symbiont luminescence is essential for cycling of escry1; this defect could be complemented by presentation of exogenous blue light. However, blue-light exposure alone did not induce cycling in nonsymbiotic animals, but addition of molecules of the symbiont cell envelope to light-exposed animals did recover significant cycling activity, showing that light acts in synergy with other symbiont features to induce cycling. While symbiont luminescence may be a character specific to rhythms of the squid-vibrio association, resident microbial partners could similarly influence well-documented daily rhythms in other systems, such as the mammalian gut. PMID:23549919